Immature regions show lower expression of <i>MAPT</i> and exon 10.

<p><b><i>A</i>,</b> Regional and temporal variation in <i>MAPT</i> expression, <b><i>B</i>,</b> exon 2 splicing, <b><i>C</i></b>, exon 3 splicing and <b><i>D</i>,</b> exon 10 splicing. *Region with p<0.05 by generalized linear model including age, region, sex and RIN score as covariates, Bonferroni’s correction for multiple comparisons. ⁺Reference region. <i>CGE</i> caudal ganglionic eminence, <i>DIE</i> diencephalon, <i>DTH</i> dorsal thalamus, <i>FC</i> frontal cerebral wall, <i>LGE</i> lateral ganglionic eminence, <i>MGE</i> medial ganglionic eminence, <i>OC</i> occipital cerebral wall, <i>PC</i> parietal cerebral wall, <i>TC</i> temporal cerebral wall, <i>URL</i> upper rhombic lip, <i>VF</i> ventral forebrain, <i>A1C</i> primary auditory cortex, <i>AMY</i> amygdala, <i>CBC</i> cerebellar cortex, <i>DFC</i> dorsolateral prefrontal cortex, <i>HIP</i> hippocampus, <i>ITC</i> inferior temporal cortex, <i>M1C</i> primary motor cortex, <i>MD</i> mediodorsal nucleus of the thalamus, <i>MFC</i> medial prefrontal cortex, <i>OFC</i> orbital prefrontal cortex, <i>S1C</i> primary sensory cortex, <i>STC</i> superior temporal cortex, <i>STR</i> striatum, <i>V1C</i> primary visual cortex, <i>VFC</i> ventrolateral prefrontal cortex, regions are defined in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195771#pone.0195771.ref027" target="_blank">27</a>].</p

qPCR on human fetal and adult cortex demonstrating shift towards 1N and 4R tau during development.

<p>Taqman primers for <b>A</b>, 0N, <b>B</b>, 1N, <b>C</b>, 2N and, <b>D</b>, 4R tau were used with normalization to GAPDH and total tau. P-values using Mann-Whitney test.</p

<i>MAPT</i> exons 2 and 10 show a rapid perinatal transition in alternative splicing in multiple datasets.

<p>p-values calculated using generalized linear models including available covariates within each dataset (Yale = age, sex, RIN; BrainSpan = age, sex; Lieber = age, sex, RIN, race; UK/NABE = age, sex, RIN).</p

0N3R is the predominant tau proteoform in human fetal cortex.

<p><b>A</b>, Immunoblots with fetal and adult brain homogenates were probed with antisera to total tau (HT7). <b>B</b>, Treatment of the fetal brain homogenates with phosphatase resolves the banding pattern to a single species with similar electrophoretic mobility to the 0N3R tau in the protein ladder. Molecular weights are indicated in kDa after each isoform.</p

Candidate cis-regulated ASM variants in phenotypically implicated SNPs.

<p>Mean number of candidate cis-regulated ASM variants in ten random LD pruned (ld<0.3) sets of candidate cis-regulated ASM SNPs that are also in high LD (LD>0.8) with a Genome Wide Association Study (GWAS) derived variant from the National Human Genome Research Institute (NHGRI) database or an eQTL in monocytes or peripheral blood monocytes (PBMCs), with accompanying standard deviations of the mean (sd).</p

Types of allele-specific methylation candidates.

<p>Plots showing number of different categories of ASM candidates within the microarray study sample population. Of the 242533 MPRs for which there were at least 6 heterozygotes within the population (left pie chart) we detected some level of ASM in at least 116045 (left pie chart, green), of these we detected ASM in at least 6 samples in 12032 MPRs (left pie chart, dark green). Of these 12032 ASM MPRs, we detected cis-regulated ASM in 9750 MPRs (right pie chart, solid blue or solid yellow), and random or stochastic ASM in 2282 MPRs (right pie chart, mixed blue and yellow). Representation of patterns of allelic-choice in ASM within the microarray study sample population. ASM allelic choice is shown at 28 ASM and 2 non-ASM MPRs for the 42 samples in our initial microarray sample population. Non-heterozygous samples (white), samples with biallelic methylation (grey), and samples with ASM (blue and yellow) with methylation at either Allele-A (yellow) or Allele-A (blue) are shown. MPRs are organized in columns to show those determined to have no ASM (first two columns), cis-regulated ASM (both for Allele-A (3rd to 11th columns) and Allele-B (15th to final columns) or random ASM (12th to 14th columns).</p

Genomic context of cis-regulated allele-specific methylation events.

<p>Illustrations showing genomic context and individual CpG methylation levels at three separate MPRs (rs10491434 (left), rs6569648 (middle) and rs943049 (right)). The chromosomal location of each amplicon is demonstrated with an ideogram. and the RefSeq genes (orange) surrounding the amplicon (red line) shown below. A section (grey box) contracts to the amplicon region itself to show the relative positions of the SNPs (black lines) and CpGs (blue lines) within the amplicons themselves (red bars); methylation levels of the alternate (grey circles) and reference (yellow circles) alleles within samples heterozygous for the SNP are graphed below each CpG. Asterixes (*) mark the CpGs illustrated within <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098464#pone-0098464-g003" target="_blank">Figure 3</a>.</p

Microarray based detection of allele-specific methylation.

<p><b>A)</b> A simplified representation of the Methyl-Sensitive Restriction Enzyme (MSRE) based Allele-Specific Methylation (ASM) assay. DNA is MSRE treated (left panels) and MSRE sites with methylated CpGs protected from digestion (upper panels, Allele-A) while its homologous chromosomal region with unmethylated CpGs are not (lower panels, Allele-B). The DNA is digested with StyI and NspI to form 200–1200 bp fragments, linkers ligated and DNA amplified to create amplicons that are hybridized to the array. Only regions with protected MSRE sites (methylated CpG) are amplified and can hybridize to show signal on the array (final panel). <b>B)</b> Bioinformatic detection of Allele-Specific Methylation (ASM) from Affymetrix SNP 6.0 arrays signals after MSRE digestion. In the scatter plot on the left, 4 different expected states after MSRE digest at a heterozygous region are compared to the typical distribution of probe intensities observed within the HapMap samples for the same MPR (here portrayed by light grey squares): biallelic methylation (dark grey circles), monoallelic A methylation (blue circles), monoallelic B methylation (yellow circles) and finally biallelic lack of methylation (red circles). The primary calling method relies on feature extraction by way of conversion of 2-dimensional A and B probe intensity data (scatter plot) from heterozygotes to log2(A/B) values and is compared against the typical log2(A/B) distribution observed for this MPR within the HapMap samples (histogram, light grey). Simply put, MPRs diverging from this distribution after MSRE treatment are called ASM. Using this method, biallelic unmethylated states have the potential to result in false positive ASM calls as any log2(A/B) value would be based on background noise, so are filtered out by removing MPRS with low total intensities (highlighted here with a red quarter-circle, for further information on how this filter was devised, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098464#pone.0098464.s008" target="_blank">Figure S8</a>).</p

Confirmation of ASM in a subset of candidate cis-regulated ASM variants.

<p>Results from microarray and next-generation bisulfite sequencing ASM assays of ten variant-containing regions. The table shows the CpG position, whether it is found within an MSRE site and the Bonferroni adjusted chi-square based p-values of the association of methylation at this CpG with either the reference of alternate alleles. The allele with the highest number percentage of methylated reads was designated the most frequently methylated allele (REF = reference, ALT = alternate) and compared to that from the microarray data; for all CpGs that were within MSRE sites and showed significant association of methylation with an allele in the sequencing assay the methylated allele matched that of the microarray assay. For more details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098464#pone.0098464.s020" target="_blank">Table S3</a>.</p

Ancient African origins of the NE1 haplogroup.

<p>(A) Models of scenarios that could lead to NE1 haplotypes observed in humans and Neandertals. The frequency of the NE1 haplogroup is depicted in red and the frequency of the nonNE1 haplogroup in yellow. The red corresponds to higher frequencies, whereas yellow corresponds to lower frequencies of the NE1 haplotypes in the population. The arrows represent the direction of possible admixture events. The left panel represents a model, under which the NE1 haplotypes admixed into Eurasian populations (Asn and Eur) after Human-Neandertal divergence. The second model, which is depicted in the central panel, is similar to the first model, except with the addition of more recent back migration of Eurasian NE1 haplotypes into Africa (Afr). The right panel shows the third model, under which the NE1 haplotypes among humans are explained by persistence of ancient African substructure. All these scenarios were based on the assumption that the NE1 haplotype occurs at high frequency or is fixed in the Neandertal population given that the available Neandertal sequences align well to the NE1 haplotype. (B) Geographical distribution of the NE1 haplogroup. We estimated the proportion of chromosomes that carry the CNVR8163.1 deletion from various sources described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003404#s4" target="_blank"><i>Materials and Methods</i></a>. The dark red portion of each circle represents the frequency of the homozygous nonNE1 genotypes, the white represents the homozygous NE1 genotypes and the light red represents the frequency of heterozygote genotypes. Note the existence of the NE1 haplotypes (i.e., as heterozygotes, <i>light</i> red) among sub-Saharan African populations (e.g., LWK and ASW) as well as the high frequency of heterozygotes (<i>light red</i>) in the European populations. (C) The pairwise distances between the African (Afr) NE1 haplotypes, the Asian (Asn) NE1 haplotypes, and the European (Eur) NE1 haplotypes, calculated using phase 1 data from the 1000 Genomes Project. p-values were calculated by the Mann-Whitney test.</p