Comment XMRV -Mouse DNA contamination -Retrovirology

[Guest guest]

RETROVIROLOGY

Mouse DNA contamination

in human tissue tested for XMRV

Mark J , Otto W Erlwein, Steve Kaye,

Weber, Oya Cingoz, Anup Patel,

Marjorie M , Wun-Jae Kim, Mongkol

Uiprasertkul, M Coffin and Myra O McClure

Retrovirology 2010, 7:108

doi:10.1186/1742-4690-7-108

````````````````

This study *Mouse DNA contamination in human

tissue tested for XMRV* by MJ et al, can

be found at: http://bit.ly/h1sz1q

~jvr

````````````````

http://bit.ly/eEqexI

Comments:

et al

flaws in study design

Gerwyn

(21 January 2011)

PA institute

This article presents a methodological critique of the

study carried out by 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 et al

appear unremarkable. The detection rate by PCR is in

line with that reported by son 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 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 son 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

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 , OW Erlwein, S Kaye, J Weber, O

Cingoz, A Patel, MM , 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 , NA , 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 son, 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-; 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