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>>>> 22 January 2011 <<<<
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RETROVIROLOGY
Mouse DNA contamination
in human tissue tested for XMRV
Mark J Robinson, Otto W Erlwein, Steve Kaye,
Jonathan Weber, Oya Cingoz, Anup Patel,
Marjorie M Walker, Wun-Jae Kim, Mongkol
Uiprasertkul, John M Coffin and Myra O McClure
Retrovirology 2010, 7:108
doi:10.1186/1742-4690-7-108
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This study *Mouse DNA contamination in human
tissue tested for XMRV* by Robinson MJ et al, can
be found at: http://bit.ly/h1sz1q
~jvr
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http://bit.ly/eEqexI
Comments:
Robinson et al
flaws in study design
Gerwyn Morris
(21 January 2011)
PA institute
This article presents a methodological critique of the
study carried out by Robinson et al (1).
In essence this article submits that the work in
question departs so profoundly from the work of
other research groups that the results must be
treated with caution.
In particular these results are at best open to
interpretation and at worst may be the product of
experimentally induced artifacts.
Initially the results of the study by Robinson et al
appear unremarkable. The detection rate by PCR is in
line with that reported by Danielson et al but some
400% lower than that achieved by IHC using XMRV
specific probes (2, 3).
This of course means that any conclusion made by
PCR alone regarding the association of XMRV and
mouse DNA is profoundly unsafe.
One must also point out that Robinson et al did not
find higher rates of XMRV infection in cancerous
versus non cancerous sequences which is a serious
departure from the results of other workers.
In fact it is such a notable departure that an
explanation is needed.
The association between XMRV and mouse DNA in
this study is imperfect whereas in a contaminant
scenario it would not be. The authors theorise that
this may be due to the inability of their PCR assay to
detect XMRV in low copy numbers.
This hypothesis was unfortunately not put to the
test by using IHC which has been shown to be far
more sensitive than PCR.
As PCR is by some considerable margin the least
sensitive method for detecting XMRV in prostate
cancer (2, 3) and chronic fatigue syndrome (4) no
association between the presence of mouse DNA
and XMRV can be safely made.
The authors expressed surprise that their PCR
approach produced gag sequences which did not
contain the 24-nt deletion characteristically present
in the XMRV sequences cloned to date, as they
expected their primers to be specific to VP62 gag.
This raises a number of issues.
Firstly, despite the authors' expectations they
provide no evidence that their primers were specific,
merely that they were capable of detecting XMRV
gag (5, 6).
The only study which has detected XMRV in prostate
cancer using primers set for unique sequences in the
XMRV genome is Schlaberg et al 2009 (2).
It is therefore something of a mystery why
researchers concerned about mouse contamination
did not engage in a literature search to establish
whether the primers used in their experiments were
in fact specific to XMRV sequences which are unique
to the virus.
This is particularly vital as if the Schlaberg primers
did not produce the same results this study would
fall.
Secondly the presence of a recombinant with a
different gag sequence is entirely consistent with the
behaviour of XMRV-related viruses in vivo and is to
expected in tumour tissue (7).
This occurs via the interaction of the nucleic acid of
the exogenous viruses and the endogenous viruses
which permanently occupy mammalian genomes.
Indeed, the clone reported by the authors which
contained a 24 base pair deletion in glycogag would
be a classic example of a viral variant caused by the
recombination of XMRV and an endogenous MLV
virus.
The authors were in a position to clarify the issue
themselves which sadly they did not.
XMRV has unique sequences in the U3 integrase and
SU region.
From the authors report they had access to
sequences or whole clones containing the
information needed to confirm whether these were
recombinants or not. It seems astonishing that they
did not provide this information in the paper.
Finally,the authors by their own admission expected
a primer sequence constructed against a region
between U5 and the 5' end of GAG to be only
capable of amplifying XMRV.
These primers were part of a specific assay
developed by Urisman et al (2006) (5) but do not
compliment any region that is unique to XMRV.
It is possible that these authors misunderstood this
essential point.
It is difficult to fathom therefore why the authors
would conclude that these expected PCR findings
were evidence of mouse contamination when a far
more parsimonious explanation was available.
Indeed, one would argue that the authors (if aware
of recent research conducted by Danielson et al
2010) (8) should have been surprised that primer
sequences that they believed were specific to XMRV
gag would have produced any product at all.
The important point to note is that at this
juncture in the study there were no results
suggestive of any mouse contamination at all.
Next we turn to the section of the study where the
authors test the ability of their PCR assays (IAP and
mtDNA) to detect mouse DNA in mouse cell lines.
The cell lines named are McCoy and RAW264.7
(RAW). It is not clear whether both cell lines were of
mouse origin.
The McCoy cell line used could have been human in
origin. If so, it would have been spiked, as the
authors stated that they amplified mouse DNA from
both cell lines.
If they were both mouse cell lines then other
considerations enter the equation.
McCoy mouse cell lines express a polytropic MLV (9)
and the RAW cell line was initially transformed by
Abelson MLV.
These cell lines have the potential of introducing
other viruses into the experimental environment.
In any event, the presence of mouse DNA in these
cells must be considered as the source of
contamination reported in this experiment.
This seems to be a parsimonious explanation as
there was no evidence of contamination prior to the
use of these cell lines.
Finally, we examine the claim made by the authors
that IAP was a much more sensitive assay than
mtDNA PCR in detecting mouse DNA in transformed
cell lines.
The normal criticism of this statement would centre
on unfounded extrapolation from in vitro to in vivo
conditions. There is however more to consider here.
The environment of transformed and immortalised
cells is one of extremely high levels of oxidative
stress (10). Mitochondrial DNA is much more prone
to oxidative damage (due to its nudity) than genomic
DNA (11).
Indeed studies have demonstrated that such cell
lines are either depleted of mtDNA (12) or that the
mitochondrial genome is highly mutated (13).
Therefore the amount of mtDNA actually recoverable
by PCR in this experiment may well have been
minimal.
Thus before any claims regarding the relative
sensitivity of the two assays were made, the
integrity of the mitochondrial DNA should have been
investigated.
Moreover while the authors demonstrated that their
IAP assay could not amplify human DNA they did not
demonstrate that it could not amplify IAP sequences
in prostate tumour tissue.
This is particularly remiss of them as IAP sequences
have only been detected in PMBCS in cases of
Sjiornes syndrome but occur at a high frequency in
the DNA of people suffering from cancer (14).
The greatest concern however relates to the known
ability of retroviruses to package IAP sequences into
their genomes.
Hence a positive response to a IAP assay
may well just indicate XMRV or PMLV
infection.
The following is a quote from A Dusty Miller
expressing his view about the IAP test in a
communication to people with ME/CFS.
*....However, unpublished results suggest that IAP
sequences might be transferred by retroviruses, so
finding IAP sequences in human samples might
simply reflect IAP transfer by the XMRV and
XMRV-like viruses we are trying to detect....*
In conclusion, the findings of this study are
inconsistent with the reports of other workers.
Given the potential for the accidental introduction of
contaminant DNA into this experiment, the
conclusions cannot be safely generalised.
Similarly the claim that IAP is superior to , or even
as reliable, as mtDNA PCR in vivo is not supported
by the data cited as alternative explanations for the
findings are readily available.
References:
1) MJ Robinson, OW Erlwein, S Kaye, J Weber, O
Cingoz, A Patel, MM Walker , W-J Kim, M
Uiprasertkul, JM Coffin and MO McClure; Mouse DNA
contamination in human tissue tested for XMRV;
Retrovirology 2010,
7:108doi:10.1186/1742-4690-7-108
2) Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh
IR (2009) XMRV is present in malignant prostatic
epithelium and is associated with prostate cancer,
especially high-grade tumors. Proc Natl Acad Sci USA
106:16351–16356
3) RS Arnold, NV Makarova, AO Osunkoya, S Suppiah,
TA Scott, NA Johnson, SM Bhosle, D Liotta, E Hunter,
FF Marshall, H Ly, RJ Molinaro, JL Blackwell, JA
Petros; XMRV Infection in Patients With Prostate
Cancer: Novel Serologic Assay and Correlation With
PCR and FISH; Urology - April 2010 (Vol. 75, Issue
4, Pages 755-761,
DOI:10.1016/j.urology.2010.01.038)
4) JA Mikovits, Y Huang, MA Pfost, VC Lombardi, DC
Bertolette, KS Hagen and FW Ruscetti; Distribution
of Xenotropic Murine Leukemia Virus-Related Virus
(XMRV) Infection in Chronic Fatigue Syndrome and
Prostate Cancer; AIDS Rev 2010; 12: 149-52
5) Urisman A, Molinaro RJ, Fischer N, Plummer SJ,
Casey G, et al. 2006 Identification of a Novel
Gammaretrovirus in Prostate Tumors of Patients
Homozygous for R462Q RNASEL Variant. PLoS Pathog
2(3): e25. doi:10.1371/journal.ppat.0020025
6) B Dong, S Kim, S Hong, J Das Gupta, K Malathi,
EA Klein, D Ganem, JL DeRisi, SA Chow, and RH
Silverman; An infectious retrovirus susceptible to an
IFN antiviral pathway from human prostate tumors;
PNAS January 30, 2007 vol. 104 no. 5 1655-1660
10.1073/pnas.0610291104
7) H van der Puttena, W Quinta, J van Raaija, ER
Maandaga, IM Verma and A Bernsa; M-MuLV-induced
leukemogenesis: Integration and structure of
recombinant proviruses in tumors; Cell, Volume 24,
Issue 3, 729-739, 1 June 1981 doi:10.1016/0092
8674(81)90099-4
8) BP Danielson, GE Ayala and JT Kimata; Detection
of Xenotropic Murine Leukemia Virus-Related Virus in
Normal and Tumor Tissue of Patients from the
Southern United States with Prostate Cancer Is
Dependent on Specific Polymerase Chain Reaction
Conditions; J Infect Dis. (2010) 202 (10):
1470-1477. doi: 10.1086/656146
9) Fong CK, Yang-Feng TL, Lerner-Tung MB;
Re-examination of the McCoy cell line for
confirmation of its mouse origin: karyotyping,
electron microscopy and reverse transcriptase assay
for endogenous retrovirus; Clin Diagn Virol. 1994
Apr;2(2):95-103.
10) RH Burdon, V Gill and C Rice-Evans; Oxidative
Stress and Tumour Cell Proliferation; Free Radical
Research 1990, Vol. 11, No. 1-3 , Pages 65-76
11) C Richter, J W Park, and B N Ames; Normal
oxidative damage to mitochondrial and nuclear DNA
is extensive; PNAS September 1, 1988 vol. 85 no. 17
6465-6467
12) J-I Hayashi, M Takemitsu and I Nonaka;
Recovery of the missing tumorigenicity in
mitochondrial DNA-less HeLa cells by introduction of
mitochondrial DNA from normal human cells;
Somatic Cell and Molecular Genetics Volume 18,
Number 2, 123-129, DOI: 10.1007/BF0123315
13) M Bakhanashvili, S Grinberg, E Bonda, AJSimon, S
Moshitch-Moshkovitz and G Rahav; p53 in
mitochondria enhances the accuracy of DNA
synthesis; Cell Death and Differentiation (2008) 15,
1865–1874; doi:10.1038/cdd.2008.122
14) W Seifarth, H Skladny, F Krieg-Schneider, A
Reichert, R Hehlmann, and C Leib-Mösch;
Retrovirus-like particles released from the human
breast cancer cell line T47-D display type B- and
C-related endogenous retroviral sequences; J Virol.
1995 October; 69(10): 6408–6416
Competing interests
None declared
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