Additional file 2: Figure S2. of Differential methylation at the RELN gene promoter in temporal cortex from autistic and typically developing post-puberal subjects

Mean ΔCt value for RELN total expression for controls (green) and for ASD brains (red) for the six pairs. Standard errors of the mean are shown. (PPTX 81.5 kb

Studies on XMRV and/or MLV-related virus in several pathologies, by country of origin of the sample.

<p>Studies were based on nested PCR or real time PCR (genomic or RT-PCR).</p>1<p>CFS, Chronic Fatigue Syndrome,</p>2<p>PBMC, Peripheral Blood Mononuclear Cells,</p>3<p>RA, Rheumatoid Arthritis,</p>4<p>ASD, Autistic Spectrum Disorder,</p>5<p>ID, Infiammatory Diseases,</p>6<p>RTI, Respiratory Tract Infections.</p>7<p>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.</p><p>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.</p

Redox and methylation pathway metabolite levels in frontal cortex of autistic and control subjects.

<p>Tissue homogenates were prepared from autistic (n = 11) and control (n = 8) subjects and thiol/thioether metabolite levels were analyzed via HPLC with electrochemical detection. Levels of homocysteine and cystathionine were significantly lower in autistic subjects (<i>p</i><0.05).</p

Redox-dependent global metabolic regulation by MS.

<p>MS activity is highly sensitive to redox status and in turn affects redox status via its influence over transsulfuration of HCY to cystathionine and cysteine. TNF-α transcription is under the epigenetic control of methylation and exerts a potent negative influence over MS mRNA levels. Changes in MS activity affect >200 methylation reactions via the SAM to SAH ratio, grouping all these reactions under the influence of redox status. Methylation regulates fundamental aspects of metabolism, including gene expression RNA processing and protein turnover. The requirement of MET t-RNA for initiation of protein synthesis underscores the central role of MS in the global coordination of metabolism.</p

Domain structure and exon composition of cobalamin-dependent MS.

<p>(<b>A</b>) MS is comprised of five domains: HCY-binding (pink), methylfolate-binding (green), cap (yellow), cobalamin-binding (red) and SAM-binding (blue). Structures from <i>E.coli </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056927#pone.0056927-Dixon1" target="_blank">[1]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056927#pone.0056927-Bandarian2" target="_blank">[3]</a> and <i>T.maritima </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056927#pone.0056927-Evans1" target="_blank">[4]</a> (PDB codes 1Q8J, 1K98 and 1MSK, respectively were used to construct a composite model). A structurally uncharacterized linker segment between the folate and cap domains is absent. (<b>B</b>) The human MS gene contains 33 exons specifying its five domains in a sequential manner. The location of PCR sequences used in this study is indicated by red and black line segments below the corresponding domains.</p

Control and autistic subject clinical details and CAP and COB mRNA values.

a<p>Autism Tissue Program identifier. All autistic samples were paired with age- and sex-matched controls.</p>b<p>Mean age (± s.d.) for control group = 20.1±6.6, for autism = 16.4±10.4; paired-t = 1.055, df = 34, <i>P</i> = 0.299.</p>c<p>Mean PMI (post-mortem interval) (± s.d.) for control group = 21.2±6.6, for autism = 23.8±10.9; paired-t = 0.8428, df = 33, <i>P</i> = 0.405. Statistical results indicate no mean differences in age or PMI. † Autism samples that were not pair-matched, but included in the mean MS mRNA analysis. <i>Italics</i> indicate cases of PDD-NOS (Pervasive Developmental Disorder Not Otherwise Specified). A.U. = arbitrary units. S.E. = status epilepticus.</p

MS mRNA in human cortex across the lifespan.

<p>Domain-specific PCR products for MS mRNA from 24 year-old (<b>A</b>) and 80 year-old (<b>B</b>) subjects. The predicted size of the PCR product is shown below each domain. Age-dependent decline in levels of MS mRNA was determined with (<b>C</b>) cobalamin-binding domain (<b>D</b>) or cap domain qRT-PCR primers. Data was fit to a two-component exponential function. (<b>E</b>) Cobalamin-binding and cap domain PCR products for subjects at 4, 49 and 84 years of age. GAPDH product for each pair of samples is show below. (<b>F</b>) Ratio of cap to cobalamin-binding qRT-PCR products for subjects <20 years <i>vs.</i> >60 years. ^* indicates significant difference from under 20 years group (p<0.0001).</p

Redox and methylation-related pathways in neurons.

<p>Neurons can utilize either astrocyte-derived or transsulfuration-derived cysteine for GSH synthesis; however, higher levels of cystathionine in human brain indicate impaired transsulfuration, increasing the importance of the former cellular uptake pathway. GSH levels are a primary determinant of cellular redox status, and oxidative stress can inhibit MS activity by multiple mechanisms, as described herein, including oxidation of Cbl(I), down-regulation of transcription, or alternative mRNA splicing with deletion of the cap domain. Oxidative stress restricts dopamine-stimulated phospholipid methylation, since it is dependent upon MS activity.</p

MS mRNA variants in human cortex.

<p>(<b>A</b>) qRT-PCR was performed using cDNA from the cortex of fetal and 76 year old subjects, with primers spanning exons 13–26, yielding three products (a–c). (<b>B</b>) Exon composition of the full-length upper band (a) and the alternatively spliced middle band lacking exons 16–18 (b) were confirmed by sequencing; the proposed composition of the lower band is also illustrated (c).</p

Control subject clinical details and CAP and COB mRNA values.

<p>Control cortex samples from age 28 weeks to 84.7 years. A.U. = arbitrary units. PMI = postmortem interval.</p