PLoS ONEplosplosonePLoS ONE1932-6203Public Library of ScienceSan Francisco, USAPONE-D-10-0591810.1371/journal.pone.0016609Research ArticleBiologyBiochemistryNucleic acidsDNADNA amplificationGeneticsMolecular geneticsMicrobiologyVirologyViral classificationRetrovirusesEmerging viral diseasesViral disease diagnosisNeuroscienceDevelopmental neuroscienceMolecular neuroscienceMedicineClinical geneticsDiagnostic medicineInfectious diseasesInfectious diseases of the nervous systemViral diseasesNeurologyDevelopmental and pediatric neurologyPediatricsDevelopmental and pediatric neurologyGenetics and GenomicsVirologyInfectious DiseasesNeurosciencePediatrics and Child HealthNeurological DisordersBiochemistryLack of Infection with XMRV or Other MLV-Related Viruses in Blood,
Post-Mortem Brains and Paternal Gametes of Autistic IndividualsXMRV or MLV-Related Viruses and Autistic
DisorderLintasCarla12GuidiFrancesco3ManziBarbara4ManciniAntonio5CuratoloPaolo4PersicoAntonio M.12*Laboratory of Molecular Psychiatry and
Neurogenetics, University Campus Bio-Medico, Rome, ItalyLaboratory of Molecular Psychiatry and
Psychiatric Genetics, Department of Experimental Neurosciences, I.R.C.C.S.
“Fondazione Santa Lucia”, Rome, ItalyInstitute of Hematology, Catholic University
of the Sacred Heart, Rome, ItalyDepartment of Child Neuropsychiatry,
University “Tor Vergata”, Rome, ItalyDepartment of Internal Medicine, Catholic
University of the Sacred Heart, Rome ItalyHarrisReubenEditorUniversity of Minnesota, United States of America* E-mail: A.Persico@unicampus.it
Conceived and designed the experiments: AMP CL. Performed the experiments: CL
FG. Analyzed the data: AMP CL. Contributed reagents/materials/analysis
tools: AMP AM PC BM. Wrote the paper: CL AMP.
The authors have declared that no competing interests exist.
2011232201162e166091122010281220102011Lintas et alThis is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original author and
source are credited.Background
Autistic spectrum disorder (ASD) is characterized by impaired language,
communication and social skills, as well as by repetitive and stereotypic
patterns of behavior. Many autistic subjects display a dysregulation of the
immune system which is compatible with an unresolved viral infection with
prenatal onset, potentially due to vertical viral transmission. Recently,
the xenotropic murine leukemia virus-related virus (XMRV) has been
implicated in chronic fatigue syndrome (CFS) and in prostate cancer by
several, though not all studies.
Methodology/Principal Findings
We assessed whether XMRV or other murine leukemia virus (MLV)-related viruses
are involved in autistic disorder. Using nested PCR targeted to
gag genomic sequences, we screened DNA samples from:
(i) peripheral blood of 102 ASD patients and 97 controls, (ii) post-mortem
brain samples of 20 ASD patients and 17 sex- and age-matched controls, (iii)
semen samples of 11 fathers of ASD children, 25 infertile individuals and 7
fertile controls. No XMRV gag DNA sequences were detected,
whereas peripheral blood samples of 3/97 (3.1%) controls were
positive for MLV.
Conclusions|Significance
No MLV-related virus was detected in blood, brain, and semen samples of ASD
patients or fathers. Hence infection with XMRV or other MLV-related viruses
is unlikely to contribute to autism pathogenesis.
This work was supported by the Italian Ministry for University, Scientific
Research and Technology (P.R.I.N. n.2006058195 and 2008BACT54), the Italian
Ministry of Health (RFPS-2007-5-640174), the Fondazione Gaetano e Mafalda Luce
(Milan, Italy), a Trailblazer Award by Autism Speaks (Princeton, N.J.), and the
Autism Research Institute (San Diego, CA). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the
manuscript.Introduction
Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder,
characterized by different levels of impairment in social interaction and
communication, as well as by stereotypies and rigid patterns of behaviour [1]. Disease onset
occurs prior to 3 years of age and its incidence is currently estimated at 1/150
live births [2]–[3]. ASD is the most heritable neuropsychiatric disorder, yet
very few cases can be solely explained on the basis of de novo
genetic mutations or cytogenetic abnormalities [4]. Vertical viral transmission
represents a non-genetic mechanism compatible with high parent-to-offspring
transmission and with low rates of disease-specific genetic abnormalities [5]. Clinically,
many ASD patients display a dysregulation of the immune system, potentially
suggestive of a prenatal-onset, unresolved viral infection [6]–[7]. Vertically transmitted viruses
should be found more frequently in the affected tissues of autistic individuals
compared to controls: based on this hypothesis we initially assessed the prevalence
of several neurotropic viruses in post-mortem brains of autistic patients and
controls, finding a significant association between ASD and polyomavirus infection
[8]. In the
present study, we focus our attention on xenotropic murine leukemia virus-related
virus (XMRV) and other xenotropic murine leukemia (MLV)-related viruses. These
retroviruses indeed represent good candidates for vertical viral transmission in
autism, because of their ability to integrate into the parental host genome and thus
undergo parent-to-child transmission. Furthermore, XMRV infection is currently a
source of serious concern in the USA for its possible link with chronic fatigue
syndrome (CFS) [9].
Using nested PCR, XMRV and MLV gag genomic sequences were sought in
the following biological samples: (a) peripheral blood mononuclear cells (PBMC)
belonging to 102 ASD patients and 97 controls, (b) post-mortem brains of 20 ASD
patients and 17 sex- and age-matched controls, and (c) semen samples belonging to 11
fathers of ASD children, 25 infertile individuals and 7 fertile controls. Our
results do not support the frequent involvement of XMRV or MLV-related viruses in
autism pathogenesis.
MethodsPatients and samples
All subjects, except for post-mortem brain donors, were recruited in Italy and
are ethnically Italian. The demographic characteristics of these samples are
summarized in Table 1.
Briefly, (a) PBMC were obtained drawing blood from ASD patients diagnosed for
any ASD (either Autistic Disorder, Asperger Disorder, or Pervasive Developmental
Disorder Not Otherwise Specified) according to DSM-IV criteria [1], and
clinically assessed as described [10]. Controls were drawn as
prescribed by family practitioner for a broad range of physical complaints
unrelated to psychiatric disorders and among nursing and medical students at
University Campus Biomedico (Rome, Italy), as described [10]; (b) frozen
post-mortem brain tissues dissected from the superior
temporal gyrus (Brodmann Areas 41/42 or 22) were obtained through the Autism
Tissue Program from the NICHD Brain & Tissue Bank (Baltimore, MD) and the
Harvard Brain Tissue Resource Center (Belmont, MA). These tissue samples largely
overlap with those employed in our previous studies [10], as this neocortical region
hosts well-documented structural and functional abnormalities in autism [11]; (c)
semen samples were provided by outpatients who underwent andrological evaluation
for infertility at the Division of Endocrinology of Catholic University of the
Sacred Heart (U.C.S.C., Rome, Italy) upon vibratory stimulation using Ferticare
Clinic (Multicept, Frederiksberg, Denmark), according to the ethical guidelines
approved by the Institutional Review Boards (IRB) of Catholic University of the
Sacred Heart and University Campus Bio-Medico (U.C.B.M.). Within 1 hour from
collection, semen specimens were separated into seminal fluid and three cellular
fractions (mobile sperm cells, sperm cells with hypomotility, and non-mobile
cells including immobile spermatozoa, immature forms, leukocytes and epithelial
cells) by centrifugation at 300 g for 30 minutes using Isolite®
(IrvineScientific, Santa Ana, CA, USA); aliquots were stored at −80°C
until DNA extraction. The consent forms signed by all individuals involved in
blood and semen collection, including parents for their children, were approved
by the Institutional Review Board of University Campus Bio-Medico
(U.C.B.M.).
10.1371/journal.pone.0016609.t001
Demographic characteristics of the samples used in this
study.
Sample type (N)
Status (N)
Mean age ± SD (year)
Sex
ASD1
(N = 20)
15.4±9.5
M∶F = 15∶5
Post-mortem brains
(N = 37)
Controls (N = 17)
17.2±8.5
M∶F = 12∶5
ASD (N = 102)
10.2±5.4
M∶F = 83∶19
PBMCs2 (N = 199)
Controls (N = 97)
50.0±15.7
M∶F = 45∶52
mobile spermatozoa
(N = 8)
hypomobile spermatozoa
(N = 8)
ASD fathers (N = 9
43.4±9.5
-
non-mobile cells (N = 9)
mobile spermatozoa
(N = 19)
Semen (N = 110)
hypomobile spermatozoa
(N = 20)
Infertile (N = 25)
35.1±6.1
-
non-mobile cells (N = 25)
mobile spermatozoa
(N = 7)
hypomobile spermatozoa
(N = 7)
Fertile (N = 7)
33.8±3.5
-
non-mobile cells (N = 7)
ASD, Autistic Spectrum Disorder.
PBMCs, Peripheral Blood Mononuclear Cell.
Nested PCR and sequencing
DNA was recovered by phenol/chloroform extraction and ethanol precipitation,
following cell digestion with proteinase K at 55°C overnight. XMRV
gag nested PCR was performed as previously described [12] with the
following modifications: approximately 80 ng of genomic DNA in 25 µl final
PCR reaction volume were used as a template for the first round PCR; 40 cycles
were done for each round of amplification. In our hands, nested PCR sensitivity
was at 10 viral copies, as in previous reports [13]. Each PCR experiment
included equal numbers of patients and controls, as well as negative controls
for the first and second round PCRs; in order to minimize the risk of
contaminations, positive controls were PCR-amplified separately and run on the
same agarose for band size determination, as in our previous studies [8].
Appropriately-sized PCR products (413 bp) were sequenced, using a CEQ8000 DNA
sequencer (Beckman-Coulter, Fullerton, CA). In order to exclude contaminations
with mouse genomic DNA, MLV positive samples were also assessed by a nested PCR
targeting mouse histone deacetylase 5 (Hdac5) and displaying the same
sensitivity as the nested PCR used to detect MLV.
Results
No MLV-related virus gag sequences were detected in 96 blood samples
and 20 post-mortem brains of ASD patients, as well as in 25 semen fractions
belonging to 9 fathers of ASD children (Table 1). Similarly, 17 control brains, and 85
semen fractions from 7 fertile and 25 infertile controls (Table 1) were negative for MLV-related
gag sequences. Three out of 97 (3.1%) peripheral blood
samples from unaffected controls were positive (Figure 1). The difference between ASD blood
samples and unaffected controls does not reach statistical significance
(Fisher's exact P-value = 0.25, n.s.). DNA sequencing and
BLAST analysis unveiled in 3 control blood samples viral gag gene
sequences displaying 100% alignment with the mouse endogenous retrovirus MLV
on chromosome 8 (GenBank Acc number: AC163617 nt 85467–85880). No mouse
genomic contamination was detected in these three positive MLV control samples by
nested PCR targeting the mouse histone deacetylase 5 (Hdac5) gene.
10.1371/journal.pone.0016609.g001
XMRV gag nested PCR on PBMC DNA, showing positive
samples in lane 5, 9 and 12.
A = autistic, C = control,
L = ladder (100 bp GeneDirex),
+ = positive control,
− = negative control.
Discussion
Our results show that infection with XMRV or other MLV-related viruses, assessed both
in the central nervous system and in blood, is not associated with ASD nor is likely
implicated in vertical viral transmission through parental gametes. We thus
replicate and largely extend a recent study reporting no association between XMRV
infection and autistic disorder [14].
A search for viruses as primary etiological agents in autism is well justified.
Congenital infection with rubella or cytomegalovirus (CMV) represents one of the
best-documented environmental factors significantly associated with ASD (for review
see [15], [16]). The
largest longitudinal study involving several hundred children prenatally exposed to
rubella virus estimates at 7.4% the rate of autism in this group, much higher
than ASD prevalence rates in the general population; risk appears especially high if
rubella infection occurs during the first 8 weeks postconception [17]–[20]. Evidence
linking prenatal CMV infection to autism is more circumstantial, but several case
reports have been published [21]–[28]. Risk estimates are essentially based on a small cohort
of 7 prenatally CMV-infected children, who displayed autistic features in 2 cases
(2/7 = 28.6%) [25].
XMRV represents an interesting candidate to potentially play a role in autism
pathogenesis. It was initially identified by PCR in approximately 10% of
prostate cancer patients [12]. It is phylogenetically related to MLV-related viruses
and displays about 90% sequence identity with MLV [12]. Recently, XMRV infection has
been strongly associated with CFS [9]. Attempts to replicate these initial results in European
and North-American cohorts of prostate cancer and CFS patients have yielded
conflicting results. In general, the association between XMRV infection and human
disease appears stronger in the USA compared to Europe (Table 2). However, also four US studies are
completely negative [13], [14], [29], [30], accounting for about two thirds of the total patient
sample recruited in North America (Table 2). The discrepancy between European and North American studies
could therefore reflect differences in PCR-based assay sensitivity rather than real
geographical differences in the prevalence of infection by XMRV or other MLV-related
viruses. In this respect, it will be important to establish and validate universal
assays, as recently proposed by the National Institutes of Health.
10.1371/journal.pone.0016609.t002
Studies on XMRV and/or MLV-related virus in several pathologies, by
country of origin of the sample.
Ref.
Country
Pathology
Tissue
Patients
Controls
Virus
[12]
USA
Prostate cancer
Prostate tissue
9/86 (10%)
-
XMRV
[9]
USA
CFS1
PBMC2
68/101(67%)
8/218(3.7%)
XMRV
[32]
USA
Prostate cancer
Prostate tissue
14/233 (6%)
2/101 (2%)
XMRV
[33]
USA
CFS
PBMC
32/37(86.5%)
3/44 (6.8%)
MLV-related
[13]
USA
CFS
PBMC
0/50 (0%)
0/97 (0%)
-
[31]
USA
Prostate cancer
Prostate tissue
32/144 (22%)
-
XMRV
[29]
USA
CFS, HIV, RA3
PBMC
0/293 (0%)
-
-
[30]
USA
Prostate cancer
Prostate tissue
0/800 (0%)
-
-
[14]
USA
ASD4
PBMC
0/134 (0%)
0/204(0%)
-
TOTAL USA7
155/1878
13/664
(8.2%)
(2%)
[14]
Italy
ASD
PBMC
0/96 (0%)
-
-
[34]
Netherlands
CFS
PBMC
0/32 (0%)
0/43 (0%)
-
[35]
UK
CFS
PBMC
0/186 (0%)
-
-
[36]
Netherlands
Prostate cancer
Prostate tissue
3/74 (4%)
-
XMRV
[37]
UK
CFS
PBMC
0/108 (0%)
-
-
[38]
Germany
Prostate cancer
Prostate tissue
1/105 (1%)
1/70 (1.4%)
XMRV
[39]
Germany
Prostate cancer
Prostate tissue
0/589 (0%)
-
-
[40]
China
CFS
PBMC, plasma
0/65 (0%)
0/85 (0%)
-
[41]
Netherlands
HIV
Seminal plasma
0/54 (0%)
-
-
[42]
France
ID5 &
others
PBMC & others
0/62 (0%)
0/99 (0%)
-
[43]
Germany
RTI6
Resp.secretions
20/267(7.4%)
2/62 (3%)
XMRV
[44]
UK
HIV and HCV
PMBC, plasma
0/232 (0%)
-
-
TOTAL REST OF THE WORLD7
24/1870
3/359
(1.3%)
(0.8%)
Studies were based on nested PCR or real time PCR (genomic or
RT-PCR).
CFS, Chronic Fatigue Syndrome,
PBMC, Peripheral Blood Mononuclear Cells,
RA, Rheumatoid Arthritis,
ASD, Autistic Spectrum Disorder,
ID, Infiammatory Diseases,
RTI, Respiratory Tract Infections.
Patients vs controls - USA:
χ2 = 30.49,
df = 1,
P = 3.35×10−8; Rest of
the world: χ2 = 0.504,
df = 1, P = 0.477, n.s.
USA vs Rest of the World - patients:
χ2 = 98.57,
df = 1,
P = 3.13×10−23;
controls: χ2 = 1.900,
df = 1, P = 0.170, n.s.
Our results, combined with those reported by Sutherfield et al [14], render XMRV contributions
to autism highly unlikely. Nonetheless we cannot exclude that MLV-related viruses
may play a role in rare cases.
The authors gratefully acknowledge all the children and families who participated in
this study. Post-mortem tissues were provided by the NICHD Brain & Tissue Bank
(Baltimore, MD) and the Harvard Brain Tissue Resource Center (Belmont, MA), through
the Autism Tissue Program (Princeton, NJ).
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