Minimal methylation classifier (MIMIC): A novel method for derivation and rapid diagnostic detection of disease-associated DNA methylation signatures

Rapid and reliable detection of disease-associated DNA methylation patterns has major potential to advance molecular diagnostics and underpin research investigations. We describe the development and validation of minimal methylation classifier (MIMIC), combining CpG signature design from genome-wide datasets, multiplex-PCR and detection by single-base extension and MALDI-TOF mass spectrometry, in a novel method to assess multi-locus DNA methylation profiles within routine clinically-applicable assays. We illustrate the application of MIMIC to successfully identify the methylation-dependent diagnostic molecular subgroups of medulloblastoma (the most common malignant childhood brain tumour), using scant/low-quality samples remaining from the most recently completed pan-European medulloblastoma clinical trial, refractory to analysis by conventional genome-wide DNA methylation analysis. Using this approach, we identify critical DNA methylation patterns from previously inaccessible cohorts, and reveal novel survival differences between the medulloblastoma disease subgroups with significant potential for clinical exploitation

Stemness of the Organ of Corti Relates to the Epigenetic Status of Sox2 Enhancers

In the adult mammalian auditory epithelium, the organ of Corti, loss of sensory hair cells results in permanent hearing loss. The underlying cause for the lack of regenerative response is the depletion of otic progenitors in the cell pool of the sensory epithelium. Here, we show that an increase in the sequence-specific methylation of the otic Sox2 enhancers NOP1 and NOP2 is correlated with a reduced self-renewal potential in vivo and in vitro; additionally, the degree of methylation of NOP1 and NOP2 is correlated with the dedifferentiation potential of postmitotic supporting cells into otic stem cells. Thus, the stemness the organ of Corti is related to the epigenetic status of the otic Sox2 enhancers. These observations validate the continued exploration of treatment strategies for dedifferentiating or reprogramming of differentiated supporting cells into progenitors to regenerate the damaged organ of Corti

Genome-Wide Scan on Total Serum IgE Levels Identifies FCER1A as Novel Susceptibility Locus

High levels of serum IgE are considered markers of parasite and helminth exposure. In addition, they are associated with allergic disorders, play a key role in anti-tumoral defence, and are crucial mediators of autoimmune diseases. Total IgE is a strongly heritable trait. In a genome-wide association study (GWAS), we tested 353,569 SNPs for association with serum IgE levels in 1,530 individuals from the population-based KORA S3/F3 study. Replication was performed in four independent population-based study samples (total n = 9,769 individuals). Functional variants in the gene encoding the alpha chain of the high affinity receptor for IgE (FCER1A) on chromosome 1q23 (rs2251746 and rs2427837) were strongly associated with total IgE levels in all cohorts with P values of 1.85×10−20 and 7.08×10−19 in a combined analysis, and in a post-hoc analysis showed additional associations with allergic sensitization (P = 7.78×10−4 and P = 1.95×10−3). The “top” SNP significantly influenced the cell surface expression of FCER1A on basophils, and genome-wide expression profiles indicated an interesting novel regulatory mechanism of FCER1A expression via GATA-2. Polymorphisms within the RAD50 gene on chromosome 5q31 were consistently associated with IgE levels (P values 6.28×10−7−4.46×10−8) and increased the risk for atopic eczema and asthma. Furthermore, STAT6 was confirmed as susceptibility locus modulating IgE levels. In this first GWAS on total IgE FCER1A was identified and replicated as new susceptibility locus at which common genetic variation influences serum IgE levels. In addition, variants within the RAD50 gene might represent additional factors within cytokine gene cluster on chromosome 5q31, emphasizing the need for further investigations in this intriguing region. Our data furthermore confirm association of STAT6 variation with serum IgE levels

SLC30A8 (ZnT8) Polymorphism is Associated with Young Age at Type 1 Diabetes Onset

It was recently shown that the major allele of the SLC30A8 (zinc transporter 8, ZnT8) single nucleotide polymorphism (SNP) rs13266634 was associated with type 2 diabetes and with reduced insulin secretion in non-diabetic relatives. Because of its role in beta-cell function, we hypothesized that this candidate SNP may confer increased susceptibility for beta-cell destruction in type 1 diabetes. We analyzed SLC30A8 genotypes in 874 patients with type 1 diabetes and 1021 control subjects. No difference in allele and genotype frequencies of the SLC30A8 SNP rs13266634 was found between patients and controls. Analysis with respect to age at type 1 diabetes onset, however, showed that patients with a diabetes onset before age 5 years had an increased prevalence of the cytosine (C) allele (risk allele, 82%) and the homozygous CC genotype (65%) compared to patients who developed type 1 diabetes after age 5 years (67% and 49%; p < 0.01) and compared to controls (69% and 48%; p < 0.03). These data suggest that genetic susceptibility for beta-cell dysfunction in the presence of autoimmunity may lead to accelerated progression and early manifestation of the disease

Characterization of Sox2 translation during OCSC isolation.

<p>(<b>A</b>,<b>B</b>) Double-labeling of Sox2 with PCNA, Bmi1, Jag1 and Hes1 in the OC at E13.5 and P4 compared to OCSCs. (<b>A</b>) Representative immunostaining images of longitudinal cryosections of the prosensory domain in the proximal cochlea duct at E13.5 (basilar membrane on top, luminal surface on the bottom). (<b>B</b>) Immature (P4) OC in mid-modiolar sections of the basal cochlea turn (medial to the left). (<b>C</b>) P4 OC-derived otic spheres after 5 DIV. Due to the requirements for the different tissue types investigated, the fixation, staining protocols and image acquisition settings were not identical (Scale Bars: A,B,C, 10 µm) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s005" target="_blank">Figure S5</a>).</p

Epigenetic, transcriptional and translational characterization of Sox2 expression during OC development.

<p>(<b>A</b>–<b>C</b>) OC during development. (<b>A</b>) Upper panel: Schematic of the sensory domain, which contains the proximal cochlea duct, showing interkinetic nuclear migration at E13.5. Sox2 expression is indicated by red nuclei. Remaining panels: marker expression at E13.5. All proliferating Ki-67-positive cells are co-labeled for Sox2. (<b>B</b>) Upper panel: schematic of the different cell types found in the maturating OC at P4. Inner hair cell (ihc, arrowhead), three outer hair cells (ohc, arrowheads) and different supporting cells: inner sulcus cells (is); interphalangeal cells (i); pillar cells (p); Deiters' cells (d); Hensen's cells (h); and Claudius cells (c). Remaining panels: marker expression at P4. The quiescence of Sox2-positive supporting cells is indicated by co-labeling with p27Kip1. (<b>C</b>) Upper panel: schematic of the different cell types found in the functional OC at P21. Remaining panels: marker expression at P21. Senescence of Sox2-positive cells is indicated by p16Ink4a expression. (<b>D</b>) RT-PCR of pluripotency marker, hair cell marker and supporting cell marker expression in the OC (E13.5, P4, P21). HPRT1 was used as the loading control. (<b>E</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, Myosin VIIa, p27Kip1 and Prox1) during OC development (E13.5, P4 and P21). The relative expression levels of P4 and P21 were compared with those at E13.5. The transcript levels were normalized to HPRT1/Ubiquitin C levels. Averages of the three independent experiments with SDs are shown (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2B</a>). Depending on the temporal expression pattern, genes were assigned to early, transition or differentiation groups (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s003" target="_blank">Figure S3</a>). (<b>F</b>,<b>G</b>) Bisulfite methylation of the Sox2 enhancers (f) (NOP1/2) and (g) (SRR1/2) during OC development (E13.5, P4, P21) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (Scale Bars: A,B,C, 10 µm).</p

Differentiation potential of OCSCs.

<p>(<b>A</b>,<b>B</b>) Methylation profiles of the otic Sox2 enhancers (<b>A</b>) NOP1 and (<b>B</b>) NOP2 in the mature OC (P21), proliferating OCSC spheres and epithelial patches differentiated from OCSC spheres (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>C</b>) Relative expression levels of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin (Myo) VIIa, p27Kip1 and Prox1) after 14 and 28 days of differentiation (n = 3) were compared with those of the proliferating OCSC spheres by qPCR. Transcript levels were normalized to TbP/Ubiquitin C levels. Shown are averages of three independent experiments (and two independent experiments for 28 days for the differentiation group) with SDs (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2D</a>). (<b>D</b>–<b>G</b>) <i>In situ</i> cell type-specific marker expression of the maturing OC (P4): Sox2 antibody (<b>F</b>) labels all supporting cells of the sensory domain (<b>G</b>), whereas S100-antibody (<b>D</b>) detects pillar and Deiters' cells only (<b>G</b>). Myosin VIIa (<b>E</b>) expression is associated with inner and outer hair cells (<b>G</b>). (<b>H</b>–<b>K</b>) OCSC-derived progeny differentiated under <i>in vitro</i> culture conditions. OCSC progeny were labeled by an EdU pulse (during the last day of 5 DIV) under proliferative culture conditions and a pulse chase after 14 DIV under differentiation-inducing culture conditions. EdU-labeling in supporting cell (Sox2, S100) (<b>H</b>) and hair cell-like (myosin VIIa) (<b>I</b>) cells. Hair cell-like cells were additionally characterized based on membrane-localized prestin (<b>J</b>) and F-actin-stained (<b>K</b>) membrane protrusions (Scale Bars: D,E,F,H,I,J,K, 10 µm) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s006" target="_blank">Figure S6</a>).</p

EGF interferes with the epigenetic regulation of Sox2 expression and affects the self-renewal potential of OCSCs.

<p>(<b>A</b>,<b>B</b>) P4 OC-derived otospheres after 5 DIV; labeling for Sox2 combined with EdU and DAPI (<b>A</b>) Otospheres grown under FGF/IGF-only conditions. (<b>B</b>) Otospheres supplemented with EGF as an additional growth factor (Scale Bars: A,B, 100 µm). (<b>C</b>) Absolute numbers of primary spheres isolated per OC with (n = 7) and without EGF (n = 8) supplementation. Data were analyzed by student's t-test and are presented as means ±SDs. (<b>D</b>) Mean diameter of the primary sphere population measured in a range from 25 to 60 µm with (n = 7) and without EGF (n = 8) supplementation. Data are presented as means ±SDs. (<b>E</b>) Methylation profiles of the otic Sox2 enhancers NOP1/2 in P21 OC, proliferating OCSCs and OCSCs supplemented with EGF (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>F</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin VIIa, p27Kip and Prox1) in standard OCSCs and in OCSCs supplemented with EGF. Relative expression levels of standard OCSCs were compared to those of OCSCs supplemented with EGF. Transcript levels were normalized to HPRT1/TbP levels. Averages of three independent experiments are shown with SDs (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2E</a>).</p

Epigenetic and transcriptional characterization of Sox2 during OCSC isolation.

<p>(<b>A</b>,<b>B</b>,<b>C</b>) Methylation profile of the Sox2 enhancers (<b>A</b>) SRR1/2, (<b>B</b>) NOP1 and (<b>C</b>) NOP2 in OCSCs as compared to the OC at P4 and E13.5 (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s004" target="_blank">Figure S4</a>). (<b>D</b>) qPCR analysis of six developmentally regulated genes (cMyc, Sox2, Atoh1, myosin (Myo) VIIa, p27Kip1 and Prox1) in OCSCs and the OC at E13.5 and P4. Relative expression levels of OCSCs and E13.5 OC were compared with those of P4 OC. Transcript levels were normalized to HPRT1/Ubiquitin C levels. Averages of three independent experiments with SDs are depicted (*p<0.05) (i.e., see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036066#pone.0036066.s008" target="_blank">Table S2C</a>).</p