Tuesday, November 19, 2013

Validation of next-generation sequencing technologies in genetic diagnosis of dementia

Brief communication


Validation of next-generation sequencing technologies in genetic diagnosis of dementia


John Becka, Alan Pittmanb, Gary Adamsona, Tracy Campbella, Joanna Kennyc, Henry Houldenb, Jon D. Rohrerd, Rohan de Silvae, Maryam Shoaib, James Uphilla, Mark Poultera, John Hardyb, Catherine J. Mummeryd, Jason D. Warrend, Jonathan M. Schottd, Nick C. Foxd, Martin N. Rossord, John Collingea, c, Simon Meada, c, Corresponding author contact information<img alt="Corresponding author contact information" src="http://origin-cdn.els-cdn.com/sd/entities/REcor.gif">, E-mail the corresponding author<img src="http://origin-cdn.els-cdn.com/sd/entities/REemail.gif" alt="E-mail the corresponding author"> a MRC Prion Unit, Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK b Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, UK c National Prion Clinic, National Hospital for Neurology and Neurosurgery, Queen Square, London, UK d Dementia Research Centre, Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London, UK e Reta Lila Weston Institute, UCL Institute of Neurology, University College London, Queen Square, London, UK




Identification of a specific genetic cause of early onset dementia (EOD) is important but can be difficult because of pleiotropy, locus heterogeneity and accessibility of gene tests. Here we assess the use of next generation sequencing (NGS) technologies as a quick, accurate and cost effective method to determine genetic diagnosis in EOD. We developed gene panel based technologies to assess 16 genes known to harbour mutations causal of dementia and combined these with PCR based assessments of the C9orf72 hexanucleotide repeat expansion and the octapeptide repeat region of PRNP. In a blinded study of 95 samples we show very high sensitivity and specificity are achievable using either Ion Torrent or MiSeq sequencing platforms. Modifications to the gene panel permit accurate detection of structural variation in APP. In 2/10 samples which had been selected because they possess a variant of uncertain significance the new technology discovered a causal mutation in genes not previously sequenced. A large proportion (23/85) of samples showed genetic variants of uncertain significance in addition to known mutations. The MRC Dementia Gene Panel and similar technologies are likely to be transformational in EOD diagnosis with a significant impact on the proportion of patients in whom a genetic cause is identified.


Keywords Dementia; Diagnosis; Neurogenetics; Genetic; Sequencing; Next-generation sequencing; NGS; Ion torrent; MiSeq


Figures and tables from this article:


Fig. 1. Histogram showing detection of APP duplications. Assessing samples for the presence of APP duplications was done using the ‘SNP-based normalisation of median depth of coverage’ tool within the NextGENe software. This tool compares the median coverage at 3 positions in each amplicon and compares this to global coverage values to generate a log2 ratio score. We then compared the mean log2 ratio of probes at APP with those at all other genes and expressed the difference in standard deviations.


Table 1. Validation panel details and Ion Torrent performance metrics


type and frequency of mutations present in genes are shown (+++ = common, ++ = occasional, + = rare; relative to other mutation types in that gene) and the actual number assessed within the validation set. The number of exons and bp screened per gene are indicated along with the average read depth achieved across 95 samples. Areas of lower coverage are shown in the far right column.


Key: bp, base pair; CNV, copy number variation; PCR, polymerase chain reaction.


Corresponding author at: MRC Prion Unit, Department of Neurodegenerative Disease, Queen Square, London WC1N3BG, UK Tel.: +44 (0)207 676 2164; fax: +44 (0)207 837 8047.


Copyright © 2014 Elsevier Inc. All rights reserved.




Wednesday, November 13, 2013


Spontaneous Generation of Infectious Prion Disease in Transgenic Mice


***These considerations enable us to hypothesize that the BSE epidemic could have begun by a random genetic mutation that was able to generate de novo infectious prions, which were included in meat and bone meal fed to cattle and then broadly expanded in the cattle population. According to this hypothesis, a key strategy for controlling BSE would involve preventing cows from consuming products from cows with spontaneous cases of BSE.***




Tuesday, May 7, 2013


Proteinopathies, a core concept for understanding and ultimately treating degenerative disorders?




Sunday, February 10, 2013


Parkinson's Disease and Alpha Synuclein: Is Parkinson's Disease a Prion-Like Disorder?




Wednesday, May 16, 2012


Alzheimer’s disease and Transmissible Spongiform Encephalopathy prion disease, Iatrogenic, what if ?


Proposal ID: 29403




Wednesday, September 21, 2011


PrioNet Canada researchers in Vancouver confirm prion-like properties in Amyotrophic Lateral Sclerosis (ALS)




Ann N Y Acad Sci. 1982;396:131-43.


Alzheimer's disease and transmissible virus dementia (Creutzfeldt-Jakob disease).


Brown P, Salazar AM, Gibbs CJ Jr, Gajdusek DC.




Ample justification exists on clinical, pathologic, and biologic grounds for considering a similar pathogenesis for AD and the spongiform virus encephalopathies. However, the crux of the comparison rests squarely on results of attempts to transmit AD to experimental animals, and these results have not as yet validated a common etiology. Investigations of the biologic similarities between AD and the spongiform virus encephalopathies proceed in several laboratories, and our own observation of inoculated animals will be continued in the hope that incubation periods for AD may be even longer than those of CJD.




CJD1/9 0185 Ref: 1M51A




Dr McGovern From: Dr A Wight Date: 5 January 1993 Copies: Dr Metters Dr Skinner Dr Pickles Dr Morris Mr Murray




1. CMO will wish to be aware that a meeting was held at DH yesterday, 4 January, to discuss the above findings. It was chaired by Professor Murray (Chairman of the MRC Co-ordinating Committee on Research in the Spongiform Encephalopathies in Man), and attended by relevant experts in the fields of Neurology, Neuropathology, molecular biology, amyloid biochemistry, and the spongiform encephalopathies, and by representatives of the MRC and AFRC. 2. Briefly, the meeting agreed that:


i) Dr Ridley et als findings of experimental induction of p amyloid in primates were valid, interesting and a significant advance in the understanding of neurodegenerative disorders;


ii) there were no immediate implications for the public health, and no further safeguards were thought to be necessary at present; and


iii) additional research was desirable, both epidemiological and at the molecular level. Possible avenues are being followed up by DH and the MRC, but the details will require further discussion. 93/01.05/4.1




BSE101/1 0136




5 NOV 1992 CMO From: Dr J S Metters DCMO 4 November 1992




1. Thank you for showing me Diana Dunstan's letter. I am glad that MRC have recognized the public sensitivity of these findings and intend to report them in their proper context. This hopefully will avoid misunderstanding and possible distortion by the media to portray the results as having more greater significance than the findings so far justify.


2. Using a highly unusual route of transmission (intra-cerebral injection) the researchers have demonstrated the transmission of a pathological process from two cases one of severe Alzheimer's disease the other of Gerstmann-Straussler disease to marmosets. However they have not demonstrated the transmission of either clinical condition as the "animals were behaving normally when killed'. As the report emphasizes the unanswered question is whether the disease condition would have revealed itself if the marmosets had lived longer. They are planning further research to see if the conditions, as opposed to the partial pathological process, is transmissible. What are the implications for public health?


3. The route of transmission is very specific and in the natural state of things highly unusual. However it could be argued that the results reveal a potential risk, in that brain tissue from these two patients has been shown to transmit a pathological process. Should therefore brain tissue from such cases be regarded as potentially infective? Pathologists, morticians, neuro surgeons and those assisting at neuro surgical procedures and others coming into contact with "raw" human brain tissue could in theory be at risk. However, on a priori grounds given the highly specific route of transmission in these experiments that risk must be negligible if the usual precautions for handling brain tissue are observed.


92/11.4/1-1 BSE101/1 0137


4. The other dimension to consider is the public reaction. To some extent the GSS case demonstrates little more than the transmission of BSE to a pig by intra-cerebral injection. If other prion diseases can be transmitted in this way it is little surprise that some pathological findings observed in GSS were also transmissible to a marmoset. But the transmission of features of Alzheimer's pathology is a different matter, given the much greater frequency of this disease and raises the unanswered question whether some cases are the result of a transmissible prion. The only tenable public line will be that "more research is required" before that hypothesis could be evaluated. The possibility on a transmissible prion remains open. In the meantime MRC needs carefully to consider the range and sequence of studies needed to follow through from the preliminary observations in these two cases. Not a particularly comfortable message, but until we know more about the causation of Alzheimer's disease the total reassurance is not practical.


JS METTERS Room 509 Richmond House Pager No: 081-884 3344 Callsign: DOH 832 121/YdeS 92/11.4/1.2




BSE101/1 0136






From: Dr J S Metters DCMO


4 November 1992






CJD1/9 0185


Ref: 1M51A




From: Dr. A Wight Date: 5 January 1993




Dr Metters Dr Skinner Dr Pickles Dr Morris Mr Murray






Wednesday, January 5, 2011




David W. Colby1,* and Stanley B. Prusiner1,2





Friday, September 3, 2010


Alzheimer's, Autism, Amyotrophic Lateral Sclerosis, Parkinson's, Prionoids, Prionpathy, Prionopathy, TSE




Saturday, November 2, 2013


APHIS Finalizes Bovine Import Regulations in Line with International Animal Health Standards while enhancing the spread of BSE TSE prion mad cow type disease around the Globe




I AGREE WITH MR. BULLARD, it’s all about trade and money, BSE TSE PRION aka mad cow type disease and sound science there from, was thrown out the window by the USDA et al that fateful day in December 23, 2003, when the USDA lost it’s ‘gold card’ of supposedly being BSE FREE, (that was and still is a sad joke though), that’s when mad cow junk science was adopted by the USDA...


see why below...kind regards, terry



Monday, November 4, 2013


*** R-CALF Bullard new BSE rule represents the abrogation of USDA’s responsibility to protect U.S. consumers and the U.S. cattle herd from the introduction of foreign animal disease




Saturday, November 2, 2013


Exploring the risks of a putative transmission of BSE to new species




Monday, October 14, 2013


Researchers estimate one in 2,000 people in the UK carry variant CJD proteins




Tuesday, October 29, 2013






Wednesday, October 09, 2013






Thursday, October 10, 2013


CJD REPORT 1994 increased risk for consumption of veal and venison and lamb




Friday, August 16, 2013


*** Creutzfeldt-Jakob disease (CJD) biannual update August 2013 U.K. and Contaminated blood products induce a highly atypical prion disease devoid of PrPres in primates




WHAT about the sporadic CJD TSE proteins ?


WE now know that some cases of sporadic CJD are linked to atypical BSE and atypical Scrapie, so why are not MORE concerned about the sporadic CJD, and all it’s sub-types $$$


Creutzfeldt-Jakob Disease CJD cases rising North America updated report August 2013


*** Creutzfeldt-Jakob Disease CJD cases rising North America with Canada seeing an extreme increase of 48% between 2008 and 2010 ***




Sunday, October 13, 2013


CJD TSE Prion Disease Cases in Texas by Year, 2003-2012




Saturday, November 2, 2013


Recommendation of the Swiss Expert Committee for Biosafety on the classification of activities using prion genes and prion protein January 2013




Saturday, November 16, 2013


Management of neurosurgical instruments and patients exposed to creutzfeldt-jakob disease 2013 December


Infect Control Hosp Epidemiol.




"different strains (of same disease), different routes of infection (of same disease), different infectivity levels (dose rate) of the (same disease) = different symptoms, different lengths of illness from 1st onset of illness to death, (of the same disease) + different cultures = different geographical locations = different strains (of same disease)...TSS"



DEEP THROAT TO TSS 2000-2001 (take these old snips of emails with how ever many grains of salt you wish. ...tss)


The most frightening thing I have read all day is the report of Gambetti's finding of a new strain of sporadic cjd in young people...Dear God, what in the name of all that is holy is that!!! If the US has different strains of scrapie.....why???? than the UK...then would the same mechanisms that make different strains of scrapie here make different strains of BSE...if the patterns are different in sheep and mice for scrapie.....could not the BSE be different in the cattle, in the mink, in the humans.......I really think the slides or tissues and everything from these young people with the new strain of sporadic cjd should be put up to be analyzed by many, many experts in cjd........bse.....scrapie Scrape the damn slide and put it into mice.....wait.....chop up the mouse brain and and spinal cord........put into some more mice.....dammit amplify the thing and start the damned research.....This is NOT rocket science...we need to use what we know and get off our butts and move....the whining about how long everything takes.....well it takes a whole lot longer if you whine for a year and then start the research!!!


Not sure where I read this but it was a recent press release or something like that: I thought I would fall out of my chair when I read about how there was no worry about infectivity from a histopath slide or tissues because they are preserved in formic acid, or formalin or formaldehyde.....for God's sake........ Ask any pathologist in the UK what the brain tissues in the formalin looks like after a year.......it is a big fat sponge...the agent continues to eat the brain ......you can't make slides anymore because the agent has never stopped........and the old slides that are stained with Hemolysin and Eosin......they get holier and holier and degenerate and continue...what you looked at 6 months ago is not there........Gambetti better be photographing every damned thing he is looking at.....


Okay, you need to know. You don't need to pass it on as nothing will come of it and there is not a damned thing anyone can do about it. Don't even hint at it as it will be denied and laughed at.......... USDA is gonna do as little as possible until there is actually a human case in the USA of the nvcjd........if you want to move this thing along and shake the earth....then we gotta get the victims families to make sure whoever is doing the autopsy is credible, trustworthy, and a saint with the courage of Joan of Arc........I am not kidding!!!! so, unless we get a human death from EXACTLY the same form with EXACTLY the same histopath lesions as seen in the UK nvcjd........forget any action........it is ALL gonna be sporadic!!!


And, if there is a case.......there is gonna be every effort to link it to international travel, international food, etc. etc. etc. etc. etc. They will go so far as to find out if a sex partner had ever traveled to the UK/europe, etc. etc. .... It is gonna be a long, lonely, dangerous twisted journey to the truth. They have all the cards, all the money, and are willing to threaten and carry out those threats....and this may be their biggest downfall...


Thanks as always for your help. (Recently had a very startling revelation from a rather senior person in government here..........knocked me out of my chair........you must keep pushing. If I was a power person....I would be demanding that there be a least a million bovine tested as soon as possible and aggressively seeking this disease. The big players are coming out of the woodwork as there is money to be made!!! In short: "FIRE AT WILL"!!! for the very dumb....who's "will"! "Will be the burden to bare if there is any coverup!"


again it was said years ago and it should be taken seriously....BSE will NEVER be found in the US! As for the BSE conference call...I think you did a great service to freedom of information and making some people feign integrity...I find it scary to see that most of the "experts" are employed by the federal government or are supported on the "teat" of federal funds. A scary picture! I hope there is a confidential panel organized by the new government to really investigate this thing.


You need to watch your back........but keep picking at them.......like a buzzard to the bone...you just may get to the truth!!! (You probably have more support than you know. Too many people are afraid to show you or let anyone else know. I have heard a few things myself... you ask the questions that everyone else is too afraid to ask.)























sporadic CJD is rising in North America, per the CJD Foundation, the incidence of sporadic CJD in the age group of 55 years and older, the incidence of sporadic cjd is 1 in 9,000. that’s a lot of spontaneous twisting of proteins, a happenstance of bad luck, a lot of bad luck going around now. ...just saying.