Sequencing Technology for Epilepsy Diagnosis

Sequencing Technology for Epilepsy Diagnosis Epilepsy: It is estimated that at many as 10% of the general populace will experience a seizure in their lifetime. (Persad et al., 2003) Epilepsy is a chronic neurological disease defined by its characteristic symptom, recurrent and spontaneous seizures. (Berg et al., 2011) (Asher Y et al., 2012) Epilepsy is a common condition with an occurrence of up to 3% in the population (Deng H et al., 2013). According to Pal D.k et al., (2010) over half of epilepsies have a genetic cause. A large body of research has gone into identifying the genetic causes and corresponding molecular mechanisms of epileptic disorders. Epilepsies have a wide variety of causes including dysfunctional ion channels (channelopathies), abnormal brain development and errors of metabolism etc. (Garofalo S et al., 2012) Research in the genetic mutations behind epilepsies is vital in further understanding the pathology of the many different conditions and developing superior treatments for the afflicted patients. Early Onset Epileptic Encephalopathy: Epileptic Encephalopathies are a large number of rare (prevalence of 1et al., 2010). The symptoms of an EE generally include severe and recurring seizures along with cognitive and developmental delay and/or deterioration. This cognitive and behavioural decline may be influenced by the seizure activity as well as the underlying mechanisms of disease (Kaiman B.A. et al., 2012). The term Epileptic Encephalopathy encompasses a wide variety of syndromes with a diverse range of genetic causes and considerable overlap with other syndromic disorders such as Autism and Mental Retardation. (Berg et al., 2011). The overlap in these syndromes and continuing progression in our understanding of EE’s has resulted in A.T. et al (2010) emphasising that the term epileptic encephalopathy should be viewed as a â€Å"concept and a description† of the wide spectrum of epileptic conditions with an encephalopathic course that are being observed in the clinic. A key diagnostic feature is that Epileptic Encephalopathies tend to occur in infancy to early childhood. While adults can suffer from epilepsy with encephalopathic features this does not tend to be as severe as the Early Onest Epileptic Encephalopathies (EOEE). Children suffering from EOEE’s rarely make it to adulthood due to their poor prognosis. Berg A.T. et al (2010). While more research is coming to light on the potential lasting damaging effects of seizures (Berg et al., 2011) what is certain is that the early onset and repetition of the severe seizures present in EOEE’s is devastating to the early development of patients and their continued growth. As the patients progress from a neonate towards childhood their symptoms will also progress. For example seizures in neonates may not be observable to a clinician as the axonal pathways are not fully myelinated yet and prevent the â€Å"surge† from reaching the motor cortex. Once the brain is fully myelinated the seizures would become observable clinically even though the onset was much earlier in the neonatal stage. This progression of the clinical presentation of epileptic seizures is mirrored in the likelihood of infants suffering from Early Infantile Epileptic Encephalopathy (Otohara syndrome) to develop into Infantile Spasms (IS) at 3-6 months in life. Patients’ suffering from IS tend to develop into Lennox-Gastaut syndrome in childhood. (Asher Y et al., 2012) Diagnosis and treatment: As seen in table 1 and table 2 there are a number of epileptic encephalopathies with a clear enough presentation to make an accurate diagnosis based on the clinical symptoms alone. However due to the rarity of these conditions and the occurrence of EOEE’s with unclear presentations (I need a %?) confirmation of a candidate mutation (gene?) is considered the gold standard when it comes to EOEE diagnosis. (Lemke J.R. et al., 2012) In Lemke J.R. (2012) it was shown that when comparing the diagnostic results of a target gene panel against a clinician’s suggested diagnosis that the gene panel results matched the suggest diagnosis in the EOEE’s with clear presentations. This accounted for 50% of the cohort. However for the remainder of the cohort with unclear EE symptoms there very few suggested diagnoses and not all of these were accurate. It has been suggested by Kay C, (2012) that the significant failure to confirm a genetic diagnosis for unclear EE’s is due to the role of de novo variants as candidates. While we may have the above lists of confirmed disease and causative gene mutations in tables 1 and 2 there are still many other EOEE’s without validated genetic causes that could be the result of de novo mutations in the patients. Considering the ever increasing list of epilepsy linked (but not validated) genes, 265 of which identified in (Lemke J.R. et al., 2012)) it is clear that the current gold standard of diagnosing EOEE’s isn’t high enough. A case study shown by Zupanc M.L., (2009) recorded the diagnostic process of a patient â€Å"Kay† that originally presented with slight head drops at 7 months old. Correlating the head drops to an epileptiform EEG (in this case a â€Å"generalised high-amplitude burst of polyspike, spike and slow wave discharges†) allowed certain conditions to be ruled out however it was highlighted that while a confident diagnosis was reached there were alternatives that cou ldn’t be ruled out. Difficulty in EOEE diagnosis can stem from the multitude of potential genetic causes available for some presentations which can lead to a clinician wandering blindly picking candidates for Sanger sequencing to identify a candidate mutation. (Lemke J.R. et al., 2012) Next generation sequencing technology: If the issue in diagnosing EOEE’s is the lack of validated genetic causes for the different presentations then the most comprehensive way to investigate the matter is to examine the EOEE genomes in comparison to controls and identify the candidate genes. Next generation sequencing technology has provided the means to do this and recent years have seen a surge of NGS based studies in EOEEs and their genetic causes. (Lemke et al., 2012) (Veeramah K.R. et al., 2012) (Veeramah K.R. et al,. 2013) The original human genome project was officially announced as completed in April 2003 (Wheeler D.A., et al. 2013). This project made use of the hierarchal shotgun sequencing method (Chial H et al., 2008). This entailed the use of bacterial artificial chromosome clones (BAC) which each housed a 100Kb fragment of DNA. Over 20,000 over these BAC clones were mapped to the human genome and the order in which these BAC clones would be aligned was the tiling path that would be followed to sequence each human chromosome. The BAC clones are further sub divided into 2 Kb fragments (appropriately sized for sequencing) and these are sub-cloned into plasmid vectors and the fragments that will undergo sequencing. The sequencing was undergone by capillary electrophoresis methods. As long as there is sufficient overlap between these sequences they can be aligned to recreate the BAC clone structure. Then using the BAC clone mapped tiling path the sequences of the BAC clones can be aligned to create a contiguous stretch of sequence that represents the human chromosome. (Mardis E.R., 2008) The advances made in this area to create the next generation sequencing technologies involve a movement away from capillary electrophoresis methods and changes in the methods of how the DNA fragments are assembled to produces the genome sequence. Whole Genome Sequencing removes the use of BAC clones and instead the genome is fragmented into different distinct size classes and placed into plasmid and fosmid subclones. By generating paired end reads and using the number of bases between these reads based on the size classes the genomes can be sequenced quicker than using BAC clones. Next generation sequencing platforms allow for massively parallel DNA sequencing and come in a variety of different set ups. As the need for BAC clones has been removed the sample preparation process is much quicker and cheaper than the method used in the original human genome project. Also while the actual run time of the next generation sequencers is longer than that of the capillary based platform the fi nal yield of reads is much higher (from 96 reads on the capillary platform to up to tens of millions on a massively parallel system). This explosion in genomic sequencing technology less than two years after the completion of the first human genome project has provided an astonishing change in the pace of genomic research. (Mardis E.R., 2008) NGS read diagrams? Next generation sequencing and epilepsy: The study of genetic diseases including the EOEE has benefitted greatly from the genome sequencing revolution. Whole genome sequencing techniques have been used to identify a de novo variant in the SCN8A gene which encodes voltage gated sodium channel pore-forming alpha-subunits. Following discovery of the variant the effects of the variant on channel function was observed and shown to implicate the gene’s involvement in EOEE and Sudden Unexpected Death in Epilepsy (SUDEP). (Veeramah K.R., et al., 2012). Targeted gene panels (next generation sequencing of a list of target genes coding regions only) has proven effective in diagnosing clear presenting EOEE and even providing a potential causative variant when no clinical diagnosis is possible. Most impressive of all was the use of whole exome sequencing (WES) to identify de novo candidate variants of relevance in 7 out of 10 children and potentially identifying 3 new genes that could be linked to EOEE (Veeramah K.R. et al., 2013 ) WES is the process of sequencing only the human exome rather than the whole genome. The exome consists of all the coding regions (exons) of the genome. Even though the exome only accounts for 1% of the whole genome it is estimated to contain 85% of disease causing mutations (Choi M et al., 2009). As of April 2013 U.S. National Human Genome Research Institute claims to sequence a whole genome at the price of $5826. The Howard Hughes Medical Institute claims to sequence the whole exome at the all-inclusive cost of $500. The Broad Institute of Massachusetts Institute of Technology estimates that whole exomes can be sequenced at four times the rate of the genome in their facility (Perkel J.M 2013). This rapid and ongoing reduction in cost is mirrored at the rate WES projects are being undertaken. With a date filter for the end of 2010 on Pubmed I performed the search â€Å"exome sequencing† and found a total of 44 articles. By the end of 2012 this 805 and by the end of 2013 the nu mber had more than doubled to 1,751. Whole exome sequencing projects have the advantage of not only being cheaper and faster than whole genome projects but also are a more comprehensive option than targeted gene panels for identifying EOEE candidate variants. While not as complete in whole genome sequencing in scope 85% covers a significant amount of data to be analysed and only leaves a 15% chance of not finding a candidate variant. It should also be noted that the exome is much easier to process and analyse while the whole genome includes non-coding areas which we are currently not really able to analyse in such a way to validate the relevant link between disease and non-coding variants (Perkel J.M 2013). Hypothesis: The question we are trying to answer with these projects is â€Å"Can whole exome sequencing detect candidate disease causing variations in early onset epileptic encephalopathy patients†? As this project involves isolated probands and one family trio it also raises the question of whether the inclusion of parent WES data allows for more rapid and accurate variant analysis. As such the end research goals are to identify likely and relevant disease candidate variations in the proband WES data and confirm their presence with Sanger sequencing The benefits of WES studies in EOEE candidate variants are numerous and important. Identifying a confirmed candidate mutation can allow an affect family to receive genetic counselling. If a clear mechanism of disease can be ascertained from the disease candidate then potentially alternative anti-epileptic therapy can be implemented based on the specific condition to improve patient prognosis even slightly. Along with this as EOEE have such a wide variety in causes and mechanisms some can respond poorly to some standard anti-epileptic drugs and lead to rapid deterioration of the patient. Identifying a genetic cause can prevent this from happening. (FIND REFERENCE) On a larger scale understanding the complex mechanisms of these devastating disorders is the only way to improve and develop treatments for these conditions and improve patient prognosis. Understanding how these rare forms of epilepsy manifest may in turn provide clues into the mechanisms of the more common forms of epilepsy . (FIND REFERENCE) As more research is beginning to highlight the role of earlier onset of seizures on the developing brain and the encephalopathic effects WES could become an effective screening tool in the future that would allow early intervention to control or prevent these devastating seizures and greatly improve patient standard of life and change EOEE to a more manageable condition. (Berg et al., 2011) References: (Persad V, Epilepsy and developmental disability) Berg AT ET al, epilepsy cognition and behaviour Asher Y Molecular bases and clinical spectrum of EIEE Scheffer I., Genetic testing in epilepsy what you should be doing Deng H Molecular biology of genetic based epilepsies D.K. Pal, A.W. Pong, W.K. Chung, Genetic evaluation and counseling for  epilepsy. Nature reviews, Neurology 6 (8) (2010) 445e453. Garofal S from genetics to genomics of epilepsy Hennekam, care for patients with ultra rare disorders Kaiman B.A. et al., A genetic diagnostic approach to infantile epileptic encephalopathies Berg AT et al Epilepsy, cognition and behaviour. Lemke J.R. Targeted next generation sequencing in epileptic disorders. Kay C whole genomes in the clinic (Veeramah K.R. et al de novo pathogenic SCN8A mutation identified (Veeramah K.R. et al, Exome sequencing reveals new causal mutations in children epilepsia (Chial H et al., 2008) DNA Sequencing Technologies Key to the Human Genome Project .(Mardis E.R., 2008) Next generation DNA sequencing methods. (Veeramah K.R., et al., 2012) de novo pathogenic SCN8A (Veeramah K.R., et al., 2013) WES 9/10 de novos (Choi M et al., 2009) Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. (Perkel J.M 2013) ScienceMag exome sequencings towards

Essay --

The Constitution permits much private religious activity in and about the public schools. Unfortunately, this aspect of constitutional law is not as well-known asui1 it should be. Some say that the Supreme Court has declared the public schools "religion-free zones" or that the law is so murky that school officials cannot know what is legally permissible. The former claim is simply wrong. And as to the latter, while there are some difficult issues, much has been settled. It is also unfortunately true that public school may not be as fully aware of this body of law as they could be. Many of the organizations are actively involved in litigation about religion in the schools. On some of the issues discussed in this essay some of the organizations have urged the courts to reach positions different than they did. Though there are signatories on both sides which have and will press for different constitutional treatments of some of the topics discussed, they all agree that the following is an accurate statement of what the law currently is. â€Å"Students have the right to pray individually or in groups or to discuss their religious views with their peers so long as they are not disruptive. Because the Establishment Clause does not apply to purely private speech, students enjoy the right to read their Bibles or other scriptures, say grace before meals, pray before tests, and discuss religion with other willing student listeners. In the classroom students have the right to pray quietly except when required to be actively engaged in school activities (e.g., students may not decide to pray just as a teacher calls on them). In informal settings, such as the cafeteria or in the halls, students may pray either audibly or silently, subj... ... the U.S. Constitution First Amendment religious liberty clauses protect private religious expression but prohibit government action to advance, coerce, or endorse religion in the public schools. The religious liberty clauses apply both to the actions of the school district and the employees of the school district. Me personally I’m atheist. Religion doesn’t bother me unless someone is forcing it to me. Being here at Wunsche High School we have a Christian Club, and last year I heard someone was trying to start a LGBT club, and the Christian club said that god didn’t like gays or others of that sexual preference. Everything now and days offends someone based on something they believe in, they are, or how they were raised. Religion is going to be a problem for as long as it is around. Until that day there isn’t much we can do about the problems that will come from it.

linguistic feature in fairy tale

Once upon a time†¦Ã¢â‚¬ ¦Ã¢â‚¬ ¦..these four little words reverberates in the souls of the children and flies their little mind into the imaginative world of magic, fairies, demons, witches, talking wolf who is always ready to capture small and pretty children in its enthralling and devilish jaws. These words squeeze the heart of the children while taking them into the world of fantasy. These words are the starting point of the fairy tales that children love to hear and enjoys every moment of it. They have a universal appeal and are timeless, though depicting the culture of the time in which they are written. The fairy tales are unique in themselves and their uniqueness lies in the distinctive and thematic appeal with which they carry themselves. Despite their typical approach there is an ample scope for creativity and inculcate within children the values that are landed from generation to generation in their innocent world. Several types of narrative styles and techniques form the part of fairy tails, yet all are distinct especially in the relationship which is developed between writer and audience. The genre lies in teaching the children and adults alike putting them into the textual journey before it starts. The basic characteristic of the fairy tale lies in the approach which the writers take in exploiting the narrative and linguistic features to the full capacity. The traditional folk tales which used to belong to oral traditions used to depend on their exceptional characteristics like prosodic phonology, intonation, the use of pauses, rhythm, the different qualities of voice, as well as on paralinguistic codes, among which gestures, mimicry, eye contact etc. Though written narratives cannot imply all these yet have developed their own independent style of narration. (Bruti, â€Å"Which narrative features†, 1999, para.17) The narrative form is encoded in a linguistic form by the use of the initial/final fixed phrases. (Bruti, â€Å"Which narrative features†, 1999, para.16). The linguistic forms further depend in the genre and on the readers for which they are written. The main role of the linguistic form is to provide information with evidences in form of tales. One of the fixed phrases lies in the tradition of the opening and closing formula, a typical example of which is â€Å"Once upon a time† and â€Å"lived happily ever after†. These expressions associate the closeness between writer and children. It creates enthusiasm among the readers and suspends their disbelief in world of fantasy in which the writer is taking them, for e.g. talking of animals, princess turning into frog unless Prince comes to kiss her and bring her to original human form etc. Besides these ritual formulas, the linguistic features include the maximum use of repetitions for making expressions and for the events to unfold. The repetition helps in the easy understanding of the texts, which reflects the mark of the oral tradition.   Other basic linguistic features are the redundant structures and they also found similarity to the oral narratives.   Ellipsis and co-ordination is the characteristic feature of redundant structure, which evolves due to the fact that writers write the stories from the children angle and organizes the information in such a manner that they brings out the actual knowledge that should be imbibed to the children. The other beauty of the fairly tale is the absence of detailed description. The physical description of characters is given more prominence then going deep into their nature and inner attributes. This is done to give semantic clarity and uniqueness. And in many fairy tales characters never change physically for e.g. If the y are young, they will ever remain young. The author can give in many cases some spatial or temporal information, for e.g. The case of hyperbolic distances like, he rode on a horse for thousands miles. Writers also have full scope for temporal deviations that means they can temporarily deviate from the original path to put the excitement and enthusiasm among the readers with the help of creating the impact of visualizations. Another feature that gives punch to the story is the impulse of the protagonists that is directly heard, and which derives him or her to comment and unfold the events taking place to create the influence on the readers. This feature is called as a Conative function, which according to dictionary means mental process or behavior directed towards action or change and including impulse, desire, volition, and striving. The tenses are used frequently in the narration. The Tenses may be either â€Å"commutative† or â€Å"narrative†: A commutative attitude comes with the involvement of the speaker, and his addressees. Narrative verb brings about detached attitude. Tenses are also classified into two categories, the retrospective, which depicts the past events, and the perspective, which takes the readers to the future. Then the verbs are also used to describe the background actions. In fairy tales, that exclusively needs to be notified is about the content or we call the theme which is ordinary i.e. it shows the reality of the life and reality of the world but its beauty lies in the context of fiction and fantasy where it is fitted which makes it more interesting and enthusiastic for the readers. It implies on the ordinary daily objects too like a mirror in which we look at ourselves. It shows the beauty of the princess but this mirror is used in a Snow White as a magic mirror having powers to show the witch her ugliness and update her about the Snow white. In many fairy tales like in the Danish fantastic tales for children, which as told in , Den fantastiske fortà ¦lling i dansk borne literatur 1967 – 2003 , there is an extensive use of figurative language, specially natural metaphors and playing with the popular phrases and clichà ©s. There is also distinguish between two traditions in fantastic tales, one is content oriented epic tradition and the other is language oriented experimental tradition.   The first trend is more communicative and the second trend included language that is easily grasped and makes an exclusive sense to the readers. There are also linguistic games and digressions, meta-linguistic elements, which more focus on the communicative style but not the content. (Centre for Children's Literature, Denmark). Fairy tales are part of the children's lives, and their world. They immerse in the children the sense of belongingness to the Supernatural elements and rejuvenate their souls and give them encouragement. Though the prince, snow white, little red ridding hood, tough princess are all fictions but are part of the children's life so the language of these fairy tales is the language of the children. References 1. Bruti Silvia, November 1999. Approaching Writing Skills through Fairy Tales, The Internet,   TESL Journal Vol. V, No. 11, (Online). Available:  Ã‚  (11 March 2007) 2. Centre for Children's Literature, Denmark. Center for bornelitteratur. (Online). Available: http://www.cfb.dk/site.aspx?p=766 (11 March 2007) 3. Godard Barbara, 1979. Crawford's Fairy Tales, SCL/ELC Volume 4.1 1979 (Online). Available:   http://www.lib.unb.ca/Texts/SCL/bin/get.cgi?directory=vol4_1/;filename=godard.htmhttp://ww (11 March 2007) 4. Hà ©bert Louis, 2006. The Functions of Language, in Louis Hà ©bert, (dir.), Signo [on-line]         Ã‚  Ã‚  Rimouski (Quebec), (Online). Available: http://www.signosemio.com. (11 March 2007) ;

Emile Berliner and the History of the Gramophone

Early attempts to design a consumer sound or music playing gadget began in 1877. That year,  Thomas Edison invented his tinfoil phonograph, which played recorded sounds from round cylinders. Unfortunately, the sound quality on the phonograph was bad and each recording only lasted for only one play. Edisons phonograph was followed by Alexander Graham Bells graphophone. The graphophone used wax cylinders, which could be played many times. However, each cylinder had to be recorded separately, making the mass reproduction of the same music or sounds impossible with the graphophone. The Gramophone and  Records On November 8, 1887, Emile Berliner, a German immigrant working in Washington D.C., patented a successful system for sound recording. Berliner was the first inventor to stop recording on cylinders and start recording on flat disks or records. The first records were made of glass. They were then made using  zinc and eventually plastic. A spiral groove with sound information was etched into the flat record. To play sounds and music, the record was rotated on the gramophone. The arm of the gramophone held a needle that read the grooves in the record by vibration and transmitted the information to the gramophone speaker. Berliners disks (records) were the first sound recordings that could be mass-produced by creating master recordings from which molds were made. From each mold, hundreds of disks were pressed. The Gramophone Company Berliner founded The Gramophone Company to mass manufacture his sound disks (records) as well as the gramophone that played them. To help promote his gramophone system, Berliner did a couple of things. First, he persuaded popular artists to record their music using his system. Two famous artists who signed early on with Berliners company were Enrico Caruso and Dame Nellie Melba. The second smart marketing move Berliner made came in 1908 when he used Francis Barrauds painting of His Masters Voice  as his companys official trademark. Berliner later sold the licensing rights to his patent for the gramophone and method of making records to the Victor Talking Machine Company (RCA), which later made the gramophone a successful product in the United States. Meanwhile, Berliner continued doing business in other countries. He founded the Berliner Gram-o-phone Company in Canada, the Deutsche Grammophon in Germany and the U.K based Gramophone Co., Ltd. Berliners legacy also lives on in his trademark, which depicts  a picture of a dog listening to his masters voice being played from a gramophone. The dogs name was Nipper. The Automatic Gramophone   Berliner worked on improving the playback machine with Elridge Johnson. Johnson patented a spring motor for the Berliner gramophone. The motor made the turntable revolve at an even speed and eliminated the need for hand cranking of the gramophone. The trademark  His Masters Voice was passed on to Johnson by Emile Berliner. Johnson began to print it on his Victor record catalogs and then on the paper labels of the disks. Soon, His Masters Voice became one of the best-known trademarks in the world and is still in use today. Work on the Telephone and the Microphone   In 1876, Berliner invented a microphone used as a telephone speech transmitter. At the U.S. Centennial Exposition, Berliner saw a Bell Company telephone demonstrated and was inspired to find ways to improve the newly invented telephone. The Bell Telephone Company was impressed with what the inventor came up with and bought Berliners microphone patent for $50,000. Some of Berliners other inventions include a radial  aircraft  engine,  a helicopter, and acoustical tiles.