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
Abstract
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.
Abstract
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
IN STRICT CONFIDENCE
Dr McGovern From: Dr A Wight Date: 5 January 1993 Copies: Dr Metters Dr
Skinner Dr Pickles Dr Morris Mr Murray
TRANSMISSION OF ALZHEIMER-TYPE PLAQUES TO PRIMATES
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
IN CONFIDENCE
5 NOV 1992 CMO From: Dr J S Metters DCMO 4 November 1992
TRANSMISSION OF ALZHEIMER TYPE PLAQUES TO PRIMATES
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
IN CONFIDENCE
CMO
From: Dr J S Metters DCMO
4 November 1992
TRANSMISSION OF ALZHEIMER TYPE PLAQUES TO PRIMATES
CJD1/9 0185
Ref: 1M51A
IN STRICT CONFIDENCE
From: Dr. A Wight Date: 5 January 1993
Copies:
Dr Metters Dr Skinner Dr Pickles Dr Morris Mr Murray
TRANSMISSION OF ALZHEIMER-TYPE PLAQUES TO PRIMATES
Wednesday, January 5, 2011
ENLARGING SPECTRUM OF PRION-LIKE DISEASES Prusiner Colby et al 2011 Prions
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
VARIANT CJD PRESENTS DIFFERENTLY IN OLDER PATIENTS
Wednesday, October 09, 2013
*** WHY THE UKBSEnvCJD ONLY THEORY IS SO POPULAR IN IT'S FALLACY,
£41,078,281 in compensation REVISED
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.)
CJD QUESTIONNAIRE USA
CJD VOICE
TSS
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.
TSS
