As known breast cancer risk factors do not accurately predict the risk of developing breast cancer, breast cancer screening is still based solely on age. At the interface between environmental exposures and gene expression, DNA methylation patterns are potential biomarkers for assessing breast cancer risk, thus allowing for implementation of personalized screening and risk-reducing strategies. We used a comprehensive high-throughput DNA methylation assay in an unprecedented study design of normal breast epithelial tissue to detect methylation changes that are causally related to breast cancer occurrence and replicated our analyses in two independent datasets of normal breast tissue and blood. We identified several methylation differences in cancer-related genes, some of which overlapped between normal breast tissue and blood and were reported in previous studies. Our findings warrant further investigation on novel biomarkers for identifying women that will benefit the most from breast cancer screening.p < 1.22 × 10−7) and 43 mapping to known genes involved in metabolic diseases with significant enrichment (p < 0.01) of pathways involving fatty acids metabolic processes. Four differentially methylated genes were detected in both site-specific and regions analyses , and three genes overlapped all three datasets . Once validated, the seven differentially methylated genes distinguishing women who developed and who did not develop a sporadic breast cancer could be used to enhance breast cancer risk-stratification, and allow implementation of targeted screening and preventive strategies that would ultimately improve breast cancer prognosis.Differential DNA methylation is a potential marker of breast cancer risk. Few studies have investigated DNA methylation changes in normal breast tissue and were largely confounded by cancer field effects. To detect methylation changes in normal breast epithelium that are causally associated with breast cancer occurrence, we used a nested case–control study design based on a prospective cohort of patients diagnosed with a primary invasive hormone receptor-positive breast cancer. Twenty patients diagnosed with a contralateral breast cancer (CBC) were matched (1:1) with 20 patients who did not develop a CBC on relevant risk factors. Differentially methylated Cytosine-phosphate-Guanines (CpGs) and regions in normal breast epithelium were identified using an epigenome-wide DNA methylation assay and robust linear regressions. Analyses were replicated in two independent sets of normal breast tissue and blood. We identified 7315 CpGs (FDR < 0.05), 52 passing strict Bonferroni correction ( As a major and increasing worldwide public health burden, breast cancer prompts the need for identification of early breast tissue molecular alterations that could be used for risk-tailored early diagnostic and effective primary prevention strategies. DNA methylation, a covalent addition of a methyl group to Cytosine-phosphate-Guanine (CpG) dinucleotides, summarizes genetic, environmental, and stochastic events that contribute to inter-individual variation in gene expression and ultimately, to variation in common complex diseases risk such as breast cancer ,2. In faDemonstrating a mechanistic link between DNA methylation patterns and breast cancer occurrence remains a considerable challenge due to the cell type specificity of DNA methylation and the cell type heterogeneity of examined tissues. Such a mechanistic link should be supported by the identification of tissue-specific DNA methylation changes in normal breast tissue prior to breast cancer occurrence . In factTo cancel out field effects secondary to the cancer environment, we used a nested case–control design to compare the normal breast tissue adjacent to primary tumors between breast cancer patients who developed a contralateral breast cancer  to those who did not develop a contralateral breast cancer. The rationale behind this design is that both breasts of the same patient presumably bear the same precancerous DNA methylation alterations that summarize the complex interplay between genetic and environmental factors associated with her individual risk of developing a primary breast cancer in either breast. To further confirm that the detected DNA methylation alterations predate a second primary breast cancer occurrence, we replicated our analyses in two independent sets of case–control pairs, in which DNA samples of normal breast tissue and blood were obtained before or at the time of a first primary breast cancer occurrence.We conducted a nested case–control study based on a cohort of 757 patients diagnosed between 2000 and 2007 with a primary invasive hormone receptor-positive and non-metastatic breast cancer at a breast cancer reference center, the “Centre des maladies du sein du CHU de Québec”. Biological characteristics of tumors were extracted from pathology reports. Demographic and clinical data collected at diagnosis were extracted from medical records and entered into a database by trained nurses and registrars. Women were eligible if they had no previous diagnosis of cancer other than non-melanoma skin cancer and did not receive any treatment prior to surgery. Using an incidence density sampling scheme, 20 patients diagnosed with a contralateral breast cancer (in situ or invasive) at least 12 months after their first breast cancer (cases) were matched (1:1) with 20 patients who did not develop a contralateral breast cancer (controls). Matching variables were the year of surgery (±2 years), age (±5 years), menopausal status, family history of breast cancer (yes/no), histologic type  of the primary tumor, human epidermal growth factor receptor 2 (HER2) status of the primary tumor, and hormone therapy (yes/no). For cases and controls, normal breast tissue was collected from breast surgery specimens. Two additional sets of case–control pairs were used to select differentially methylated sites that predate a second primary breast cancer occurrence. The first set consisted of four breast cancer patients diagnosed with a first invasive hormone receptor-positive and non-metastatic breast cancer (cases) drawn from the same cohort described above, and four women diagnosed with a benign breast lesion (controls) drawn from the tissue biobank of the “Centre des maladies du sein du CHU de Québec”. Women were matched (1:1) for the year of surgery (±2 years) and age (±5 years). For cases and controls, normal breast tissue was collected from breast surgery specimens prior to any other treatment. The second set consisted of six women with high mammographic density (>65%) who eventually developed an invasive breast cancer (cases), and six women (controls) with low mammographic density (<15%) who did not develop a breast cancer by the time their matched case developed a breast cancer. These women were drawn from a cohort of 737 women who attended a mammography screening at the “Clinique radiologique Audet”  between February and December 2001 . AnthropAll participants provided written informed consent. The study protocol was reviewed and approved by the research ethics committee of the CHU de Québec-Université Laval Research Center. The data that support the findings of this study are available upon reasonable request from the corresponding author (C.D.). The data are not publicly available due to legal restrictions to respect research participant privacy and consent.The design of the main analysis aimed at identifying differentially methylated CpGs sites in normal breast epithelium that are causally associated with breast cancer occurrence, i.e., while canceling out cancer field effects, by comparing normal breast tissue of two groups of patients both exposed to similar cancer field effects. The rationale behind this design is that once cancer field effect has been canceled out from the affected breast, normal breast epithelium both from the affected breast and the non-affected contralateral breast harbor the same epigenetic marks for each individual patient, because both breasts have been exposed to the same genetic and environmental factors. In other words, this study design is comparable to having sampled the unaffected breast  and followed-up patients for the development of a primary breast cancer in this unaffected contralateral breast. Matching for factors that are known to be associated with contralateral breast cancer occurrence ensured that the setting is similar to obtaining normal breast tissue from women who had never had a previous breast cancer and comparing women who develop a primary breast cancer to those who do not develop a primary breast cancer. Beyond a simple validation of our findings in the exact same study design, we decided to select differentially methylated CpGs sites that replicate in a more traditional (but less robust because of confounding by cancer field effects) study design. In the secondary dataset #1, we compared women who have developed a first primary breast cancer to women who had not developed a first primary breast cancer, which is the setting “artificially” created by the robust design of the main dataset.Finally, the rationale behind the choice of the secondary dataset #2 was to select those differentially methylated CpGs that could also be detected in blood-derived DNA, i.e., those methylation marks that may have been induced early during development and propagated soma-wide and that could be useful as non-invasive biomarkers for breast cancer screening. Here, we compared women who developed a primary breast cancer with women who did not develop a primary breast cancer, by using blood samples prospectively collected several years before breast cancer occurrence in cases. These women had a mammography screening at time of blood collection, and those who developed breast cancer during follow-up had higher breast density at baseline than those who did not develop a breast cancer during follow-up (breast density is a known risk factor for breast cancer occurrence).Thus, our three datasets compared women who developed a breast cancer (first primary or second primary breast cancer) to women who did not develop breast cancer, using three different strategies.For breast tissue samples, normal breast epithelium located at least 1.0 cm from the primary tumor of cases and controls was identified on corresponding hematoxylin-eosin (H&E) stained slides. Ten to fifteen cores of 1.0 mm with at least 75% epithelial cells content were extracted from formalin-fixed paraffin-embedded (FFPE) tissue blocks and were used to build a tissue microarray (TMA) block for each patient. TMA blocks were serially sectioned at 10.0 μm. H&E-stained histologic tissue sections were verified for cellular content in the first, every 10th, and in the last section. A column-based method for DNA extraction from TMA sections of each patient was performed using GeneRead DNA FFPE deparaffinization solution  and the QIAamp DNA FFPE kit  for subsequent extraction steps. Deparaffinization was done twice to ensure complete paraffin elimination and proteinase K was incubated at 56 °C in ATL buffer for three days with 20 µL of proteinase K added each 24 h.For blood samples, DNA was extracted from buffy coats using the Gentra Puregene DNA extraction kit  following the manufacturer’s protocol.Quantification of DNA methylation was carried out at McGill University and Génome Québec Innovation Centre  using the Infinium Human Methylation 450K (HM450k) BeadChip , after bisulfite conversion, Infinium FFPE quality control, and DNA restoration, according to the manufacturer’s instructions. The HM450k has been extensively validated and provides reliable coverage of 485,512 CpG sites across 99% of RefSeq genes and 96% of CpG islands in the human genome [Raw methylation data preprocessing and statistical analyses were performed using R software version 3.6.2  and Biocp-value > 0.01, probes overlapping a CpG site or single-base extension of the measured methylation loci, cross-reactive probes [Data from IDAT files were read using the minfi package . Qualitye probes , probes e probes , and proe probes .The data-driven separate normalization method from the wateRmelon package was used for background adjustment and between-array normalization . The regGlobal methylation differences between cases and controls for each set were compared using the Wilcoxon signed-rank test and mean beta-values, both globally and by CpG island and gene spatial distribution.p-values  < 0.05 were selected for functional annotations  and were analyzed using the gometh function of the missMethyl package [p-value < 1.22 × 10−7) in the main analysis and those replicated in the secondary analyses were further compared to the CPGA SAGE database, using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 to select differentially methylated genes for which significant methylation changes are consistent with their differential expression in breast cancer.Probe-wise differential methylation analysis using M-values was performed with the limma package robust linear models. Robust empirical Bayes method was used to generate moderated paired t-test statistics and associated p-values for each CpG site. For blood samples, white blood cell type proportions were estimated using the method described by Houseman et al.  within t package . CpG sitp-value < 0.05, maximum difference > 0.05, and containing at least two or more CpG sites were selected. Genes identified by the region approach were compared to those identified by the individual CpG site approach.To identify concordant differentially methylated regions of several consecutive CpG sites (distance to the next consecutive site less than 1000 nucleotides), differential methylation analysis of regions with Benjamini and Hochberg correction for multiple comparisons was performed using the DMRcate package . RegionsThe 40 patients included in the main analysis were aged between 33 and 69 years , were mainly postmenopausal, and 75% reported a family history of breast cancer. They had stage I to III estrogen receptor (ER)-positive invasive breast carcinomas. All received hormone therapy and 70% received chemotherapy. Their characteristics, according to case or control status, are summarized in The eight patients included in the secondary analysis of normal breast tissue were aged between 47 and 66 years , were mainly postmenopausal, and 63% reported a family history of breast cancer. Their characteristics, according to case or control status, are summarized in The twelve women included in the secondary analysis of blood samples were aged between 38 and 53 years , were all premenopausal, and 16.7% reported a family history of breast cancer. Their characteristics, according to case or control status, are summarized in There was no global methylation difference between cases and controls, both for average methylation across all included CpGs sites and when CpGs were grouped by island location or by gene region .q-values < 0.05, of which 954 CpGs had a q-value < 0.01 . These 52 CpGs were predominantly located in CpGs islands and gene body regions, and 56% were hypomethylated. Forty-three of them were mapped to known reference genes involved in metabolic diseases. Pathway analysis of these 43 genes identified six enriched pathways  involving fatty acids metabolic processes , GLS2, and NCOR1, harbored significant methylation changes consistent with their differential expression in breast cancer.Of the 409,741 included CpGs in the main analysis of normal breast tissue, 7315 CpGs were differentially methylated at FDR e < 0.01 . Functioe < 0.01 . Fifty-trocesses . One genrocesses , and 15 p-value < 0.05, of which 15,505 CpGs had a nominal p-value < 0.01. None were differentially methylated at an FDR q-value < 0.05 , of which three were mapped to known reference genes  at a nominal e < 0.05 . Of these < 0.05 . Of thesp-value = 0.0003) involving negative regulation of cholesterol/sterols biosynthetic processes  associated with breast cancer risk in the main analysis of normal breast tissue . Pathwayrocesses . Ninety-pG sites . The 20 LHX2 was included in three DMRs, TFAP2B in two DMRs, JAKMIP1 in one DMR, and SEPT9 in one DMR. Of the 43 differentially methylated genes associated with breast cancer risk in the individual CpG sites analysis of normal breast tissue main analysis, q-value < 0.05 and 52 CpG sites at the strict Bonferroni nominal p-value < 1.22 × 10−7, of which 43 were mapped to known genes involved in metabolic diseases. Pathway analysis of these 43 distinct genes identified six enriched pathways  involving fatty acids metabolic processes.The present study aimed at identifying normal breast tissue methylation patterns that may predispose to breast cancer development, using a robust study design unprecedented in previous breast cancer epigenome-wide association studies. To cancel out field effects, our main nested case–control analysis compared normal breast tissue adjacent to primary tumors of breast cancer patients who developed and those who did not develop a contralateral breast cancer. We identified 7315 individual CpG sites with an FDR LHX2, harbored significant methylation changes at two different CpG positions, while 15 genes harbored significant methylation changes consistent with their differential expression in breast cancer. Of these, LHX2, TFAP2B, JAKMIP1, and SEPT9 were also included in significantly differentially methylated regions. The LHX2 gene codes for the LIM homeobox 2 protein, a transcription factor downstream of p63 and NF-κB, and upstream of Wnt/β-catenin, Bmp, and Shh [TFAP2B gene codes for the transcription factor AP-2 beta, a sequence-specific DNA-binding protein that has been recognized as an oncogene that mediates cancer cell proliferation, apoptosis, invasion, and migration via the COX-2 signaling pathway in vitro and in vivo [JAKMIP1 gene codes for the Janus kinase and microtubule interacting protein 1 and has been shown to be highly expressed in tumor samples, where it enhances the proliferation of cancer cells [SEPT9 gene codes for Septin 9, a protein involved in cytokinesis and cell cycle control that has been implicated in early breast cancer development [SEPT9 gene methylation has been detected in breast cancer tissue [One gene,  and Shh , that ha and Shh . This ge and Shh . The TFA in vivo . TFAP2B  in vivo  and has  in vivo . The JAKer cells . Its uprer cells . The SEPelopment . The SEPr tissue .POM121L2, KCNQ1, and CLEC4C, harbored significant methylation changes consistent with their differential expression in breast cancer. The POM121L2 gene codes for POM121 transmembrane nucleoporin like 2, which has been shown to be upregulated in triple negative breast cancer [KCNQ1 codes for the potassium voltage-gated channel subfamily Q member 1, which has been shown to play important physiological roles in the mammary epithelium [CLEC4C codes for a lectin-type cell surface receptor that may play a role in antigen capturing by dendritic cells, inflammation, and immune response, and has been shown to be upregulated in triple negative breast cancer [To further detect DNA methylation alterations that predate a second primary breast cancer occurrence, we used two independent sets of case–control pairs in which DNA samples of normal breast tissue and blood were obtained before a second breast cancer occurrence. Out of the 7315 individual CpG sites identified in the main nested case–control analysis, six CpG sites were also differentially methylated with the same direction of association in both secondary sets’ analyses, of which five mapped to known reference genes. Of these, three genes, namely t cancer . KCNQ1 cithelium  and has ithelium ,35. CLECt cancer .ZSWIM6), cg22731164 (GPR176), and cg18726036 (FKBP5) in a study of blood DNA methylation from the Sister Study [(DLX2-AS1) in a study of genetically predicted DNA methylation of patients from the Breast Cancer Association Consortium [Many epigenome-wide studies have investigated the association between DNA methylation and breast cancer risk using blood-derived DNA and the HM450k BeadChip, while as few as three studies measured breast tissue DNA methylation [er Study , and cg0nsortium , all of nsortium ,40,41,42Taken together, our findings support the hypothesis that detectable methylation differences in cancer-related genes in normal breast tissue predate the occurrence of breast cancer. Some of these methylation changes were also detectable in blood DNA, suggesting that these methylation changes may have been induced early during development and propagated soma-wide ,43, and a priori, up-to-date, and recommended data preprocessing methods and workflow, which prevent inflation of the false-positive rate resulting from data-driven selection of preprocessing methods. In addition, we conducted both site-specific and DMR analyses, and we replicated the analyses in two independent datasets. The main limitation of the study is the relatively small sample size, which could have limited the detection of genuine methylation differences . However, by using the appropriate data preprocessing methods coupled with the doubly robust statistical modeling approach, which minimizes the risk of false-negative rate, we were able to detect more differentially methylated CpG sites than larger studies [Using an original and novel study design, we were able to assess methylation changes in normal breast epithelial tissue while minimizing the risk of confounding by cancer field effects. The main strengths include the use of conventional epidemiological approaches to control for selection bias (nested case–control design) and confounding bias (matching for breast cancer risk and prognostic factors), two important drawbacks in previous epigenome-wide DNA methylation studies of breast cancer . We used studies .While robust and promising, our results need to be validated in other populations and with other DNA methylation measurement methods. Epigenome-wide DNA methylation methods are particularly suitable for hypothesis generation as they capture the dynamics of several sites simultaneously across the entire genome, thus being less prone to bias than candidate gene methylation studies . The nexLHX2, TFAP2B, JAKMIP1, and SEPT9) in the main analysis of normal breast tissue, and three genes overlapping the main analysis and two independent datasets of normal breast tissue and blood . No significant global methylation differences were observed between cases and controls in any of the three datasets. Once validated, our identified genes could be used to enhance risk stratification for prevention of breast cancer and for developing new strategies for primary breast cancer prevention and treatment.We identified four breast cancer risk-related genes that are differentially methylated in both site-specific and DMR analyses (


Thyroid hormones are critical for mammalian brain development. Thus, chemicals that can affect thyroid hormone signaling during pregnancy are of great concern. Perfluorohexane sulfonate (PFHxS) is a widespread environmental contaminant found in human serum, breastmilk, and other tissues, capable of lowering serum thyroxine (T4) in rats. Here, we investigated its effects on the thyroid system and neurodevelopment following maternal exposure from early gestation through lactation , alone or in combination with a mixture of 12 environmentally relevant endocrine disrupting compounds (EDmix). PFHxS lowered thyroid hormone levels in both dams and offspring in a dose-dependent manner, but did not change TSH levels, weight, histology, or expression of marker genes of the thyroid gland. No evidence of thyroid hormone-mediated neurobehavioral disruption in offspring was observed. Since human brain development appear very sensitive to low T4 levels, we maintain that PFHxS is of potential concern to human health. It is our view that current rodent models are not sufficiently sensitive to detect adverse neurodevelopmental effects of maternal and perinatal hypothyroxinemia and that we need to develop more sensitive brain-based markers or measurable metrics of thyroid hormone-dependent perturbations in brain development. In humans, insufficient thyroid hormone levels at critical times in neurodevelopment can induce long-term intellectual and behavioral impairments4. These outcomes can have serious consequences for both the affected individuals and society as a whole, including increases in healthcare expenses and a reduction in lifetime income6.Thyroid hormones are critical for normal brain development. The spatiotemporal action of thyroid hormones is essential for orchestrating the developmental processes of neurogenesis, migration, synaptogenesis, and myelination12.The thyroid hormone system can be affected by insufficient iodide intakes, exogenous substances such as chemicals and drugs, or by some diseases. Congenital hypothyroidism and iodine deficiency, conditions that severely reduce thyroid hormones during early development, result in severe mental retardation. However, even subclinical maternal T4 deficiency (hypothyroxinemia) can impact neurodevelopment and IQ of the child20. Yet, with regard to low-grade thyroid hormone disruption, a relationship with adverse effects on brain development is less obvious27. Still, environmental chemicals can perturb the thyroid axis through a variety of mechanisms, often inducing more subtle changes in hormonal status than seen with anti-thyroid drugs such as propylthiouracil (PTU) and methimazole (MMI)29. This means that, to better protect humans against thyroid disruption, we need to allow for more sensitive assays in rodent toxicity testing. This, because establishing a relationship between modest degrees of serum thyroid hormone decrements and neurodevelopmental sequelae is crucial for the regulation of thyroid disrupting chemicals, as current legislative regulation of thyroid disruptors relies on linking a change in serum hormone levels to a consequential adverse effect.In rodents, severe thyroid hormone deprivation clearly impairs brain development30. PFHxS is found in drinking water supplies31 and has repeatedly been one of the top three PFASs to which humans are environmentally exposed34, yet its toxicological effects remain poorly characterized37. We recently reported that developmental exposure to perfluorohexane sulfonate (PFHxS) caused a marked reduction in total serum thyroxine (T4) levels in both rat dams and their offspring36. Herein, to address the possible health impacts of PFHxS exposure, we investigated the effects on the thyroid hormone system and potential neurobehavioral consequences  constitute a class of environmental chemicals that is frequently reported to cause thyroid hormone disruption36. For completeness, some of these data are included here as percent of control . A greater reduction to 84% of controls was seen on PD 22 in the 25 mg/kg PFHxS group , Nkx2.1, Tpo, Tshr, Pax8 and Dio1)(data not shown). Thus, TSH levels, thyroid gland weight, histopathology and gene expression indicate a lack of thyroid gland perturbation in response to exposure or as a compensatory reaction to decreases in serum T4.On PD 22, no statistically significant effects on dam thyroid gland weights were seen, apart from a slight reduction in response to the EDmix . Thyroid gland histopathology was not significantly affected by PFHxS exposure, and neither were the expression levels of six gene transcripts involved in thyroid hormone synthesis and regulation (Slc5a5 (NIS), Nkx2.1, Tpo, and Tshr) (data not shown).In the PD 22 female offspring, dose-dependent decrements in thyroid gland weight were observed Fig. . These d36. Here we extended these examinations to histological assessment of liver sections from female offspring (PD 17), but found no morphological changes in liver that could explain the increases in liver weight (data not shown). Minimal midzonal microvesicular vacuolation was observed, but the change was evenly distributed between exposed and control animals. Neither did histological assessment of dam livers (PD 22) reveal any differences between control and high dose animals (25 mg/kg PFHxS) (data not shown).We previously reported increased liver weight in both male and female offspring exposed to PFHxS and/or EDmix during development, first evident in females at exposures of 5 mg/kg PFHxS40. No clear evidence for altered expression due to thyroid hormone insufficiency was observed for Agt, Col11a2, Gjb6, Hr, Ntf3, or Pvalb  the effect was qualitatively consistent with previous reports44.The expression of eight gene transcripts was assessed in male offspring on PD 16. These target genes were selected from a suite of genes previously shown to be significantly reduced in the parietal cortex of 14 days old rat pups in response to developmental exposure to the antithyroid drug, PTUHabituation, the expected decline in motor activity over the 30 min test period, was observed in offspring at all ages . Similarly, the expected sexually dimorphic pattern was seen in the adult controls, with higher activity levels in females compared to males. Treatment-related effects on motor activity levels at the three ages were minimal and did not correlate with PFHxS dose or thyroid hormone levels during development. Overall, effects suggest disrupted sexual differentiation of the male and female brain, although the results do not clearly indicate whether this mainly arise from an effect in a specific sex or from altered development of both sexes. The most notable changes at the three ages are described below.In pre-puberty (PD 27) no sexual dimorphism in motor activity levels was observed  and no significant effect of PFHxS exposure was seen in either male or female offspring Fig. . When diIn young adulthood (PD 115), no significant effects of PFHxS exposure were seen in either sex, when overall activity count was assessed Fig. . In contWhen the animals were tested for the third time (~PD 340), the expected sexually dimorphic pattern of higher activity levels in females remainedFig. . Yet, inBoth male and female offspring showed improved performance during the three-week testing period, manifested as decreased time to complete the test and a reduction of errors made Fig. . While s36 and moderate reductions in T3 in both dams and offspring. Significant dose-dependent declines were evident in dams within 7 days, increasing in magnitude with continued exposures.PFHxS exposure caused marked reductions in total T445. Thyrotropin-releasing hormone (TRH) activates the pituitary to release TSH, which binds to TSH receptors on the thyroid gland, upregulating synthesis and release of thyroid hormone into the blood. Indications of TSH-mediated thyroid gland activation include increases in thyroid gland weight, histological changes, and upregulation of certain gene transcripts48. The pattern of effects induced by the anti-thyroid drugs MMI and PTU conform to this classic view of the hypothalamic-pituitary-thyroid (HPT) axis. There are however a number of reports showing how PCBs, PBDEs and other PFASs exhibit another pattern of effects, corresponding to the one seen in the present study, i.e. clear reductions in circulating levels of T4 and T3 without an accompanying rise in TSH and activation of the HPT axis53. Consistent, with observations with another PFAS chemical, PFOS, reductions in serum T4 levels without HPT axis activation have been seen after both long-term adult exposure50 and developmental exposure of dams and pups55.Despite the marked decreases in circulating thyroid hormone levels, no significant effects on TSH levels were found in dams (PD 22) or in offspring (PD 16 and PD 17). Based on the classic models of thyroid physiology and regulatory feedback loops, diminished levels of circulating T4 are accompanied by increases in TSH35. Unfortunately, TSH levels and thyroid gland weights were not reported35. Butenhoff et al. (2009) also reported clear sex differences in toxicokinetics, where parental females exposed to 3 and 10 mg/kg/day exhibited low serum PFHxS concentrations. In contrast, parental males had higher serum PFHxS levels, a different PFHxS distribution to organs, and showed clear signs of toxicity. The sex difference in adults is attributed to differential high expression of organic anion transporters in the kidneys of female rats that ensure efficient excretion of PFHxS into the urine58. Hence, male rats are exposed to much higher serum PFHxS levels than females. In agreement with our findings in rats, PFHxS showed no effect on TSH, thyroid gland weights nor histopathology in mouse dams and pups after exposure to somewhat lower internal PFHxS levels37. Unfortunately, T3 and T4 were not measured by Chang et al.37.A previous report investigating PFHxS found histopathological changes in thyroid gland of adult male rats after 42 days of PFHxS exposure at 3 and 10 mg/kg, suggestive of HPT axis activation, but with no effects in dams or offspring following developmental exposures35, although the long-term and high dose effects of PFHxS remain uncertain, it is clear that this class of chemicals does not activate the HPT axis in a manner similar to PTU/MMI, countering our understanding of HPT axis function. Despite a significant number of studies61, the mechanistic understanding of the phenomenon of marked reductions in circulating total T4 levels without a compensatory response within the HPT axis have yet to be determined, and the consequences of this form of thyroid hormone perturbation on brain development and function requires further study.Based on the results on PFHxS exposed male ratsin vitro assays suggest that these compounds bind to the serum distributor proteins TTR and albumin64. This MoA has been explored in vivo for the structurally similar perfluorinated compound PFOS (having a C-8 chain instead of the C-6 chain in PFHxS). Chang et al. (2008) showed a dramatic and transient rise in free serum T4 in the hours following administration of PFOS (presumably due to displacement of T4 from distributor proteins), followed by an increased turnover and loss of thyroid hormone through urine and feces65. Consistent with our findings, total T4 remained low without the expected rise in TSH. Here, we hypothesize that prolonged PFHxS exposure causes a steady-state T4 level at 30% of control levels with equilibrium between distributor-protein bound T4 and distributor-protein bound PFHxS.The mode of action (MoA) by which PFAS in general, and PFHxS specifically reduces serum T4 has not been fully elucidated, but the predominant knowledge revealed by 50. However, the effects in the thyroid and liver were only evident at 5 mg PFOS/L drinking water and above, whereas clear effects on serum T4 were already detectable at the lower dose of 1.7 mg/L50. This disparity in dose level suggests that MoAs other than metabolism could underlie decrements in serum hormones. In our study, we did not observe an increase in Dio1 expression in thyroid glands, whereas liver deiodinases and glucuronidases were not investigated.Another possible thyroid MoA of PFAS is induction of liver enzymes (as a separate mode of action or secondary to TTR binding). PFOS exposure for 91 days decreased T4 levels, presumably through increased thyroidal conversion of T4 to T3 by deiodinase type 1 (increased Dio1 mRNA was reported) and increased degradation of T4 in liver by induction of thyroid hormone glucuronidation via liver enzyme UGT1A137 and PFOS-exposed rats66. With regard to other potential sites of chemical interference with thyroid hormone synthesis and signaling, PFHxS was a positive inhibitor of NIS in vitro67 but had no or minor activity at the thyroid receptor level69. Similarly, PFOS did not affect pituitary function in rats65. To our knowledge, the effects of PFHxS on thyroperoxidase (TPO) inhibition or peripheral deiodinases have yet to be investigated.Perfluorinated compounds may also augment hormone clearance through other pathways in the liver. Upregulation of the hepatic nuclear receptors peroxisome proliferator-activated receptor alpha (PPARα), constitutive androstane receptor (CAR) and pregnane X receptor (PXR) has been reported in PFHxS-exposed miceWhile PFHxS caused dose-dependent reductions in serum T4 in both dams and offspring, these reductions were not associated with changes in cortical gene expression or any neurobehavioral effects measured in offspring.et al., 2018 identified a suite of thyroid hormone responsive genes in neonatal cortex and suggested that they may serve as potential molecular readouts of TH action in the developing brain40. In this and other reports, expression levels of these genes were altered in a dose-dependent manner under conditions of thyroid hormone insufficiency71 or T3 activation72. On PD 16, where PFHxS-exposed pups exhibited reduced serum T4 levels, we hypothesized correlations between serum and brain T4 levels76, and conversely decreased thyroid hormone-mediated gene expression in the cortex. This however, was not the case. There are several possible explanations for this result. In PFHxS pups, T4 levels were reduced by 45%, a reduction similar to the effect size necessary to observe effects on brain T3, T4 and brain gene expression in a similar study with the potent anti-thyroid drug PTU (obtained at an exposure of 1 ppm PTU on PD 14)40. Therefore, the 45% reduction in serum T4 by PFHxS may have been at the threshold for imparting a molecular signal in the brain. After PTU exposure at the lowest dose of 1 ppm, the reduction in gene expression was modest but its significance strengthened by the severe and unambiguous reductions at higher doses40. As PFHxS is less potent than PTU in terms of the maximal degree of serum T4 reduction obtained, the modest effects by PFHxS exposure on only two of the 8 cortex genes remain equivocal. Additionally, severe and sustained reductions in serum/brain thyroid hormone inclusive of fetal and earlier postnatal periods that disrupt development trajectories may be necessary to provide a foundation for the gene expression changes in cortex observed with PTU. Comparable degrees of hormone perturbation may not have been achieved by PFHxS during critical fetal and early neonatal time windows. The findings suggest that snapshots of serum T4 levels, although important indicators of the potential of thyroid mediated neurotoxicity, may not serve as direct surrogates for brain hormone concentrations and consequent thyroid action under all xenobiotic exposure conditions. Compensatory mechanisms in the PD 16 pup brain, including the upregulation of local deiodinases and thyroid hormone transporters, may be sufficient in PFHxS exposed pups to maintain local levels of brain hormones and support normal transcription of cortical gene targets investigated in this study. Alternatively, genes other than the ones examined here or in different brain regions and at different developmental times may have been affected by PFHxS exposure.Targeted gene expression profiling was used to interrogate potential effects of thyroid hormone disruption on the developing rat neocortex. Based on a developmental PTU model, O’Shaughnessy PFHxS exposure did not alter motor activity assessed at three time-points in the post-weanling rat or impair performance in the radial arm maze. Does this suggest that PFHxS is without effect on brain development? Perhaps, but it may be the case that the current metrics applied in rodent models are not sufficiently sensitive to detect adverse neurodevelopmental effects of maternal and perinatal hypothyroxinemia.13. In models where severe hormone deficiencies are induced by PTU, MMI or a thyroidectomy, there is a clear association between reductions in serum T4 during the perinatal period and a wide range of behavioral impairments20. In contrast, studies investigating developmental hypothyroxinemia induced by other substances, have not shown behavioral effects clearly correlated to T427. We hypothesize that some of the discrepancy between PTU and environmental chemicals arises from differences in timing, severity and duration of the induced fetal and postnatal T4 suppression. These factors are highly influenced by toxicokinetics, complicated under conditions of pregnancy and lactation, and likely MoA of chemical interference with thyroid signaling. The data on fetal and brain T4 supply indicate that PTU very effectively restricts fetal serum and brain T470 while not much is known for most other compounds. Possibly, rodent behavior models detect effects after only very marked and sustained reductions in fetal and neonatal thyroid hormones as seen in studies where the lowest doses of PTU did not induce significant deficits in neurobehavior77. Yet, there was impaired synaptic plasticity and structural defects in the forebrain20 indicating that neurotoxicity can exist despite the lack of behavioral readout, supporting the view that simple behavioral tests typically employed in rodent toxicology studies lack sensitivity to detect consequences from modest serum T4 reductions. Still, in humans, adverse effects on neurobehavioral indices are found in children of women within low-normal range of circulating serum T479 indicating that chemically-induced thyroid hormone disruption during pregnancy is a potential risk to human brain development. Functional parameters such as IQ and language development are commonly affected, but these behavioral domains are not readily assessed in rodent models. Documented ‘functional’ impairment however is a critical component of the regulatory framework within the EU. Identification of an endocrine disrupting chemical in the EU is based on the World Health Organization (WHO) definition: an exogenous substance that alters the function of the endocrine system to cause adverse health effects in an intact organism or its progeny3. For regulatory actions to be taken on thyroid disrupting chemicals in the EU system, evidence of reduction in circulating hormone must be associated with an adverse  effect and be plausibly linked to a MoA relevant to the thyroid system. Therefore, there is a critical need to develop reliable and sensitive assays to detect neurological impairments associated with developmental thyroid hormone disruption.We chose the behavioral tests based on previous work using PTU to disrupt thyroid action, in which functional impairments were observed in adult offspring despite full recovery of thyroid hormone status36, where weak signs of sex hormone disruption  were induced by the EDmix and 0.05 mg/kg PFHxS. The subtle and non-monotonic nature of the response raises the possibility that these observations could be the result of sampling error. However, if valid, effects at such a low dose are of heightened concern for human health and warrant further study. The effects occurred at a dose far lower than what would be tested in traditional developmental toxicology studies and consequently could be missed in standard regulatory testing scenarios. Additional studies targeting the dose-response relationships for effects on sex differentiation and behavior could serve to expand our understanding of these elusive effects and improve our ability to safeguard human health in the future.Despite the relative absence of thyroid hormone-dependent effects of PFHxS on functional outcomes in this study, we did discover effects of the lowest dose of PFHxS suggestive of altered sexual differentiation of the brain, and on learning and memory. These effects were restricted to the lowest dose of 0.05 mg/kg PFHxS and were uncoupled from the serum thyroid hormone reductions evident at higher doses. The EDmix itself significantly increased activity in both PD 27 and PD 115 males, i.e. making these males more “female-like” in their behavior, supporting our previous studiesPFHxS reduced T3 and T4 in pregnant dams and their progeny but did not appear to activate the HPT axis at doses up to 25 mg/kg bw/day. The thyroid hormone disruptions were not correlated with effects on motor activity or learning and memory; rather the findings suggest that the primary effect of low doses of PFHxS may be to disrupt sexual differentiation of the brain.Human data show that T4 reductions during pregnancy and in newborns can impair brain development and cause reductions in a child’s IQ. Despite PFHxS-induced reductions in circulating T4 levels, no evidence of thyroid hormone-mediated neurobehavioral disruption in offspring was observed. However, we maintain that a significant reduction in T4 alone should warrant concern and that the metrics currently applied in rodent models are not sufficiently sensitive to detect adverse neurodevelopmental effects of maternal and perinatal hypothyroxinemia. But until we have discovered more sensitive brain-based markers or measurable metrics of thyroid hormone-dependent perturbations in brain development, we cannot with certainty determine whether or not PFHxS-mediated reductions in circulating thyroid hormone levels can adversely affect brain development.36, and herein depicted in Fig. 39. However, acetaminophen (paracetamol) that was included in the prior study by Christiansen et al. (2012), was not included in the EDmix of this study. The resulting total EDmix dose was 32.11 mg/kg bw/day36. The EDmix contained some chemicals capable of disrupting thyroid hormone levels, although given at doses well below reported No Observed Adverse Effect Levels (NOAELs). Combined in a mixture, however, these chemicals may have contributed to the observed effect by the EDmix on dam T4 levels36.The full animal study design has been previously reportedPregnant dams were received on gestation day (GD) 3 of pregnancy (the day following overnight mating was designated GD 1) and dams were dosed once daily by oral gavage from GD 7 to postnatal day (PD) 22, but not on the day of delivery. The expected day of delivery (GD 23) was termed PD 1 for all pups. Hence, the age of the pups was related to time of mating rather than day of birth. Animals were housed in a controlled environment: Reversed light/dark cycles of 12 hours (light from 9 pm–9 am), humidity 55 ± 5%, temperature at 21 ± 1 °C, and ventilation changing air ten times per hour. All animals were fed ad libitum on a standard diet with Altromin 1314  and were provided ad libitum acidified tap water  in PSU bottles (84-ACBTO702SU Tecniplast).At weaning (PD 22), one male and one female pup (when available) from each litter were weaned and housed pairwise with an animal of the same sex and group . This cohort of weaned offspring was subjected to behavioral assessment of motor activity levels at three ages, and for the control and PFHxS-only groups, learning and memory were tested in the radial arm maze (see below for descriptions). The study was terminated when the males were approximately 12 months and the females 13 months old.The animal experiments were carried out at the DTU National Food Institute facilities . Ethical approval was given by the Danish Animal Experiments Inspectorate. The authorization number given is 2015-15-0201-00553 C3. The experiments were overseen by the National Food Institute’s in-house Animal Welfare Committee for animal care and use. All methods in this study were performed in accordance with relevant guidelines and regulations.Necropsy of one male and one female (when available) per litter took place on PD 16 and PD 17, respectively, and again on PD 22, when dams were also included (see below).2/O2, decapitated, and trunk blood collected for hormone analysis (see below). Livers were excised and saved for histopathological examination (control and 25-Px groups)(see below). Thyroid glands were excised, weighed and saved for histopathology (control and PFHxS-only groups)(see below) in block 1–3. Thyroid glands from block 4 were stored in RNAlater at −80 °C for analysis of gene transcripts (control and PFHxS-only groups).On PD 22 dams were weighed, anesthetized with CO2/O2, decapitated, and trunk blood collected for hormone analysis. Livers from females were excised and saved for histopathology (PD 17)(see below). Male pup thyroid glands were excised with a piece of the thyroid cartilage and saved for histopathology (see below). Thyroid glands from females were excised, weighed, and those from block 1 and 2 were stored in RNAlater at −80 °C for gene transcript analysis (control and PFHxS-only groups).On PD 16/17 and PD 22 pups were weighed, anesthetized with COOn GD 15, tongue blood was taken from the dams without anesthesia. Male pups on PD 16 and female pups on PD 17 were euthanized by decapitation and trunk blood collected. Dam trunk blood was also collected at weaning of the pups on PD 22. The blood was collected in heparinized microcentrifuge tubes (GD 15) or in heparinized 4- or 10 ml vacutainer tubes for the pup blood and the dam blood, respectively. Blood was kept on ice until centrifugation for 10 min. at 4 °C and 4000 rounds per minute (rpm) and plasma was stored at −80 °C. T3 levels were analyzed by electrochemiluminescence-immunoassay (ECLIA) – photoncount (Cobas 8000 E-modul) at the Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark. For the dams, 15 samples from each exposure group (20 controls) were sampled randomly for analysis. For the pups, each litter was represented by either a male or female pup .Thyroid Stimulating Hormone (TSH) was measured in rat plasma with the Milliplex MAP Rat Thyroid Magnetic Bead Panel Luminex  according to the instructions of the manufacturer on randomly chosen samples from male (n = 11–13) and female pups (n = 5–7) PD 16/17  and dams PD 22 (control and PFHxS-groups only).A predefined (thin and uniform) standardized slice of the liver from the left lateral and right medial lobes were fixed in 10% formalin, processed, embedded in paraffin, and 5μm sections were stained with haematoxylin and eosin (H&E) following standard procedures. Livers from dams (PD 22) and female offspring (PD 17) were evaluated, blinded to exposure groups, for hepatocellular hypertrophy and vacuolation (macro- and microvesicular) in the control and high-dose (25-Px) groups.Sections of thyroid glands from dams (PD 22)  and male offspring (PD 16) (control and 25-Px groups) were fixed in 10% formalin, embedded in paraffin, and 5 μm sections were stained with H&E. Histological examination of thyroid glands was conducted blinded to exposure groups to evaluate signs of pathological changes as detailed below.A: No remarksB: Mild alterations with few irregular follicles or few follicles with multi-layered epithelium, increased cellularity.C: Follicular epithelial hypertrophy or hyperplasia, irregular cells or follicles, multi-layered follicular epithelium, papillary projections in follicular lumens and/or small peripheral follicles localized on the rim of the gland.Thyroid glands from dams and male pups were assigned to one of the following three categories:Slc5a5 (NIS), Nkx2.1, Tpo, Tshr, Pax8, and Dio1. Transcripts evaluated in cortical samples included: Agt, Col11a2, Gjb6, Hr, Itih3, Klf9, Ntf3, and Pvalb. B2m and Gapdh were used as reference genes for thyroid gland and cerebral cortex, respectively. Data were analyzed by the 2−∆∆Ct method80.Both lobes of the thyroid gland from female PD 17 offspring were stored in RNAlater (Ambion AM7021) at −80 °C. until RNA extraction. Thyroid glands from PD 22 dams were snap frozen in liquid nitrogen and stored at −80 °C until analysis of one lobe. An oblique slab of anterior to lateral cortex was collected from PD 16 male offspring and stored in RNAlater at −80 °C until RNA extraction. RNA was extracted with TRI Reagent (Sigma T9424) according to manufacturer’s protocol and glycogen added to facilitate precipitation of the thyroid gland RNA. RNA pellets were resuspended in nuclease-free water, DNased  and quantitated with Quant-iT RiboGreen RNA assay kit (Life Technologies R11490e). 2 µg RNA was reverse transcribed (Life Technologies High Capacity cDNA Reverse Transcription Kit 4374966) and the resulting cDNA was amplified using TaqMan Gene Expression Assays (Life Technologies) and TaqMan Gene Expression PCR Master Mix (Life Technologies 4369510) according to manufacturer’s protocol. Amplification was performed on an ABI model 7900HT sequence detection system in duplicates. TaqMan probes are detailed in Table One male and one female pup from each litter were assessed for motor activity on PD 27. The same animals were tested as young adults (PD 115) and again later in life (PD 342/336).The animals were placed individually in clean, empty cages with a flat rack lid. Cages were placed in activity boxes with photocells recording horizontal activity for 30 min. Six animals were tested simultaneously and, in all test rounds, both male and female offspring were represented. Neither food nor water were supplied during the testing. Movement was registered as interruption of a photobeam and data automatically collected by a computer in an adjoining room. The data collection started after a 10 second habituation and continued for 10 periods of 3 minutes amounting to a total of 30 minutes of observation. Motor activity was quantified as activity counts (disruption of adjacent photobeams) and break counts . Activity counts allowed determination of whether the animal was moving about the cage and not just continuously interrupting the same beam. Total activity during the 30 minutes was used as a measure of general activity and compared across groups for each sex as well as within groups for sex difference. To assess habituation, activity counts were divided into three periods, initial (1–9 min), middle (10–21 min), and last period (22–30 min).Adult offspring from the PFHxS-only groups  were tested in the radial arm maze. One male and one female from each litter were assessed, females at 4–5 months and males at 8–9 months of age. This age difference was necessitated by the large number of animals to be tested and the limitations of the capacity of the maze system. All animals had previously been tested on motor activity. Due to technical difficulties, the radial arm maze was reduced to 7 arms. In the pre-testing week the animals were fed only 15 g of chow a day in the afternoon and hand-fed a raw peanut  to accustom them to the taste of the food reward used in the maze. Following the pre-testing week, the animals received 1 trial/day in the radial arm maze. Animals were introduced to the central space of the maze and allowed to explore until they had entered and traversed to the end of each arm or until 10 mins had elapsed. Testing was performed for five consecutive days, followed by a 2-day break for a total of three weeks. Analysis was performed based on calculations of “time”  and errors . Data analysis included measures of total time and errors for each animal. To assess learning over time a mean per day during week 1, 2 and 3 was calculated.et al., 2018). The full model included the PFHxS and EDmix exposures with their interactions parameterized as indicator variables. This supported a determination as to whether (and to what magnitude) exposure to the EDmix changed the effects of PFHxS exposure alone across the different exposure groups. Because many samples were considered in the full model, much smaller effect changes could be identified than would be possible using a simple pairwise comparison. The model was tested by a main factor for EDmix and dose-dependent interaction terms to account for non-parallel dose-response patterns between PFHxS and PFHxS + EDmix.Data with normal distribution and homogeneity of variance were analyzed using analysis of variance (ANOVA). Data were transformed if normal distribution and homogeneity of variance was not present. When relevant, body weight was included as a covariate in the analyses, e.g. when testing terminal organ weights. Histology results were analyzed with Fisher’s test when analyzing 2 groups and Chi square tests for more than two groups . To avoid litter effects no more than one pup per litter was included in any analysis. Where an overall significant treatment effect was observed, Dunnett’s test was used for two-tailed comparison with the alpha level for statistical significance always set to 5%. Exposure groups were tested against the vehicle control by testing the PFHxS exposed groups against the control, all PFHxS + EDmix groups against the EDmix group and by using a t-test to compare the EDmix group against the control group. In addition, the study design with dose response exposure to PFHxS and the addition of groups exposed to both PFHxS and the EDmix allowed for construction of a statistical model integrating all exposures and control groups into one full linear model the “full model” (described in detail in Ramhøj SAS Enterprise Guide 4.3 (2010), SAS Institute Inc, Cary, NC 27513, USA, was used for statistical analyses.Supplementary Information.


Nine of these sites were taken forward in a replication study, performed in an independent case–control (n = 186) cohort using pyrosequencing. Sites in or close to FKBP5, AIM2 (2 probes), UHRF1, KCNAB2  successfully replicated. The blood-based DNA methylation signal was tissue- and disease-specific, in that the replicated probe signals were unchanged in case–control studies using sCJD frontal-cortex (n = 84), blood samples from patients with Alzheimer’s disease, and from inherited and acquired prion diseases. Machine learning algorithms using blood DNA methylation array profiles accurately distinguished sCJD patients and controls. Finally, we identified sites whose methylation levels associated with prolonged survival in sCJD patients. Altogether, this study has identified a peripheral DNA methylation signature of sCJD with a variety of potential biomarker applications.Prion diseases are fatal and transmissible neurodegenerative disorders caused by the misfolding and aggregation of prion protein. Although recent studies have implicated epigenetic variation in common neurodegenerative disorders, no study has yet explored their role in human prion diseases. Here we profiled genome-wide blood DNA methylation in the most common human prion disease, sporadic Creutzfeldt–Jakob disease (sCJD). Our case–control study (The online version of this article (10.1007/s00401-020-02224-9) contains supplementary material, which is available to authorized users. Human prion diseases are typically rapidly progressive neurodegenerative conditions associated with misfolding of prion protein (PrP) . They inPRNP), particularly the polymorphism at codon 129 in the PRNP gene where methionine  or valine is encoded and both homozygous genotypes are at increased risk of disease , FKBP5, METTL9, UHRF1, KCNAB2, MIR1977). Two more sites were used as controls: one within the prion gene (PRNP) and another within ANK1, a gene whose hypermethylation has consistently been reported in Alzheimer’s disease (AD)  were associated with a longer survival in patients with sCJD . When taking the genotype of PRNP codon 129 into account, this analysis revealed that the level of DNA methylation at two sites (UHRF1 and KIAA0513) refines the prediction on disease duration for MM patients only , cg17641710 (GNAI2), and cg17714703 (UHRF1). This effect was seen in the “Low” methylation group of cg05343106 (DNAJB13) and cg17641710 (GNAI2), and “High” methylation group of cg17714703 (UHRF1). Furthermore, combining together these three loci methylation profiles provided greater accuracy than each locus in predicting clinical outcomes. The distribution of beta values from these three DMPs was not significantly different from normality, thus we transformed them into z-scores . Average z-scores were correlated  with disease duration . Figure PRNP genotype at codon 129. Together, these results demonstrate a potential utility of profiling DNA methylation in whole blood from patients with sCJD: these profiles can help discriminate sCJD patients from sex- and age-matched healthy controls and may help predict disease duration.Finally, we evaluated the association between DNA methylation levels and survival. In our cohort, as expected, codon 129 of the prion gene Whilst DNA methylation has become increasingly studied in the context of neurodegenerative disorders, DNA methylation profiles have not yet been investigated in human prion diseases. Here, we performed a case–control study to analyse the relationship between DNA methylation and sporadic CJD using 405 peripheral blood samples from patients and controls. The discovery study used a genome-wide 450 K Illumina BeadChip array, with replication using a second technology, pyrosequencing. Seven of nine sites that we identified successfully replicated. We went on to show that these effects were tissue and disease specific and could be exploited for diagnostic and biomarker purposes. Overall, we highlight the potential of DNA methylation array profiling of peripheral blood for a rare and serious neurodegenerative disorder.Like GWAS, the more recently developed EWAS is subject to biases, in particular variability in the measured methylome differences between case and controls groups unrelated to the pathobiology of interest. Strategies have been developed to measure and correct for biases, in particular we found Houseman’s reference-based algorithm that corrects for alterations in cell composition proved successful in reducing the study-wide inflation , 61. In We questioned the relevance of the magnitude of the effect size we report here compared with the effects seen in cancerous tissues . The milConcordance of DNA methylation signatures between blood and brain has been reported in ALS . HoweverAIM2 promoter correlates with disease severity. AIM2 is a key component of the inflammasome pathway, a component of the innate immune system that drives the production of the inflammatory cytokine interleukin-1β (IL-1β) in response to microbial and nonmicrobial signals [FKBP5 DNA methylation and elevated cortisol levels. FKBP5 binds to glucocorticoid receptors and modulates glucocorticoid sensitivity. Epigenetic regulation of FKBP5 and its consequences on patient’s behaviour is well documented: accelerated age-related decreases in FKBP5 methylation are associated with childhood trauma and depressive phenotypes [FKBP5 have been found in patients suffering from post-traumatic stress disorders (PTSD) and major depressive syndromes [FKBP5 and the hypothalamic pituitary adrenal (HPA) axis (where FKBP5 plays a major role), it could be that the HPA axis provides a link between the pathology in the brain and the periphery.Although blood-based DNA methylation might not be generally considered as a surrogate for brain tissues, DNA methylation profiles detected in peripheral tissues might remain useful as biomarkers . Our fin signals . In yeas signals , 32. How signals . Second,enotypes , whilst yndromes . Such chFKBP5 in prion diseases. Work from the Soto group showed that FK506, a calcineurin (CaN) inhibitor which binds to FKBP5, substantially decreased the severity of clinical signs in mice presenting symptoms of prion disease. In the same study, the authors report that FK506, also known as tacrolimus, reduces brain degeneration and increases survival [C expression due to a defect in the translocation of PrPC into the endoplasmic reticulum with subsequent degradation by the proteasome [FKBP5 DNA methylation decreases along the life span; this age-related decrease is not confounded by blood cell type heterogeneity and occurs in purified immune cell subtypes [FKBP5 upregulation promotes NFkB-related peripheral inflammation and chemotaxis. The role played by FKBP5 in inflammation and PTSD resonates with the function of another gene found to be differentially methylated in sCJD blood: AIM2. Indeed, PTSD cases are more likely to have high levels of C-reactive protein (CRP), a widely used measure of peripheral inflammation, and this association is mediated by methylation at the AIM2 locus [Several lines of evidence support the implication of survival . Anothersurvival . Stocki oteasome . More reoteasome . Finallysubtypes . The samM2 locus .We acknowledge several limitations of this study. Having independent replication and a larger sample size of DNA methylation data would further increase the generalizability of the classifiers identified in this study. Additionally, since methylation values can change throughout the life span, it will be insightful to evaluate the methylation signature over the course of the disease, longitudinally. In the long-term, we seek to determine when the alterations to DNA methylation patterns begin to show relative to the onset of prion diseases, and whether robust and stable DNA methylation patterns can predict the onset of disease in those at high risk of the disorder. We also aim at investigating DNA methylation profiles in first-degree relatives of the sCJD patients. We were underpowered to confidently determine whether some changes discovered in sCJD were shared with other very rare prion diseases, so it remains possible our findings associate with multiple aetiological groups. Further, whilst we compared to non-prion disease neurodegenerative disorders, these affect more specific brain functions in early stages, and are less aggressive in progression. These superficial disease-related differences may contribute to observed differences in blood DNA methylation, not necessarily only pathways specific to prion disease pathobiology. Finally, the brain samples were not selected because of PrP abnormalities, yet all cases had misfolded PrP immunoreactivity confirmed on neuropathological examination.What are the functional consequences of these mild changes for cellular malfunction and disease? Future work will help investigate the mechanisms that underlie role of the selected CpG on sCJD establishment and progression. In line with this, our study can only provide correlative evidence for a blood-based sCJD-specific DNA methylation signature that robustly discriminates sCJD patients from controls, and other types of prion disease and AD patients. EWAS studies do not allow us to infer whether the DNA methylation observed represents a cause or consequence of sporadic CJD. Further investigations and functional studies will be required to understand better the contribution of epigenetic changes to sCJD.KIAA0513 and UHRF1) refined survival information driven by PRNP codon 129 genotype is informative, particularly for individuals most at risk. PRNP codon 129 methionine homozygosity is associated with shorter disease duration in sCJD [Finally, our results suggest that DNA methylation profiling could be of use to refine sCJD disease management. We show that a data-driven machine learning approach using DNA methylation profiles accurately distinguishes sCJD patients from healthy individuals using peripheral blood. To date, our study is the first to report an assay that is capable of identifying sCJD patients from a blood sample, and that discriminates between sCJD and AD. Moreover, it is also the first study to suggest that DNA methylation could be used as a blood biomarker in human prion diseases. In line with this, we additionally demonstrated that a risk score based on DNA methylation of three identified sites predicts disease duration. Work from Zhang et al. recently demonstrated that DNA methylation age acceleration associated with ALS survival . The fin in sCJD  and therRegardless of the underlying mechanisms, our results demonstrate that non-protein-mediated information about sCJD disease status is present in blood and suggest that mapping such DNA methylation patterns alterations, with future independent replication, might be of use for testing and counselling. Future work will unravel whether DNA methylation is also altered in acquired prion diseases, which involve peripheral pathogenesis. This study is meaningful in providing new avenues for understanding sporadic CJD disease mechanisms and identifying biomarkers which complement existing clinical signals in the periphery.Additional file1 (PDF 26864 kb)Additional file2 (CSV 59199 kb)Additional file3 (XLSX 18 kb)Additional file4 (XLSX 14 kb)Additional file5 (XLSX 12 kb)Additional file6 (XLSX 14 kb)Below is the link to the electronic supplementary material.


In this review, we offer a comprehensive description of 3D printing techniques commonly used to develop immunosensors, arrays, and microfluidic arrays. In addition, recent applications utilizing 3D printing in immunosensors integrated with different signal transduction strategies are described. These applications include electrochemical, chemiluminescent (CL), and electrochemiluminescent (ECL) 3D-printed immunosensors. Finally, we discuss current challenges and limitations associated with available 3D printing technology and future directions of this field.Detecting cancer at an early stage of disease progression promises better treatment outcomes and longer lifespans for cancer survivors. Research has been directed towards the development of accessible and highly sensitive cancer diagnostic tools, many of which rely on protein biomarkers and biomarker panels which are overexpressed in body fluids and associated with different types of cancer. Protein biomarker detection for point-of-care (POC) use requires the development of sensitive, noninvasive liquid biopsy cancer diagnostics that overcome the limitations and low sensitivities associated with current dependence upon imaging and invasive biopsies. Among many endeavors to produce user-friendly, semi-automated, and sensitive protein biomarker sensors, 3D printing is rapidly becoming an important contemporary tool for achieving these goals. Supported by the widely available selection of affordable desktop 3D printers and diverse printing options, 3D printing is becoming a standard tool for developing low-cost immunosensors that can also be used to make final commercial products. In the last few years, 3D printing platforms have been used to produce complex sensor devices with high resolution, tailored towards researchers’ and clinicians’ needs and limited only by their imagination. Unlike traditional subtractive manufacturing, 3D printing, also known as  Cancer is one of the leading causes of death worldwide. Globally, it was responsible for approximately 9.6 million deaths in 2018 ,4. BecauThe crucial need for effective cancer screening and accessible diagnostic tools has driven research endeavors utilizing cancer biomarkers in liquid biopsy samples like blood, urine, and saliva. Analyzing cancer markers in liquid biopsy samples overcome hurdles associated with solid tumor biopsy as it provides a rapid, precise, and non-invasive assay strategy ,8, and dSeveral analytical strategies have been adapted for development of ultrasensitive detection of protein biomarkers associated with different types of cancer. Immunoassay format is the most commonly used technique for analysis due to the inherent specificity associated with the use of antibodies as molecular-recognition agents ,11. Immu® technology) by Quanterix®  . D. D45]. D2 laser beam to melt powder beads into metallic, plastic or ceramic layers. The laser beam scans through the powder bead to print one layer, then printing platform moves down allowing addition of a fresh layer of the powder beads which is then sintered and bind to the previous layer , it suffers from low resolution (80 µm) and complex printing setup 2+ (RuPVP) and ruthenium tris  dichlororuthenium-(II) hexahydrate are among the most commonly used substrates . F. F93]. FChemiluminescence (CL) can also be used to determine the concentration of an analyte by measurement of the luminescence intensity initiated by a chemical reaction . Unlike 2O2 in biological fluids and plant extracts.We reported the first non-polydimethylsiloxane (PDMS), transparent 3D printed device with channels, detection chamber and reagent mixer A integraHybrid sensors with capability to integrate multiple components play a crucial role in developing newer technologies and deliver better user interaction. Ability to design and fabricate such integrated hybrid sensors during the proof of concept stage provides greater advantage than using simple off the shelf commercial sensors. Such an attempt requires addressing unique challenges, and 3D printing with ability to make complex shapes and sizes, using multi-material, nano-material integration and 3 dimensional structures using conductive inks/materials will provide enhanced sensing capabilities. Most importantly, 3D printing aids in consolidating a working prototype of such hybrid sensors with less creation time and cost.Integration of 3D printed electrodes made of materials composed of metal-based inks and conductive materials have played a key role in development of novel electrochemical and electro-optical micro devices and they offer several benefits 1. Rapid prototyping—full realization of a manufactured prototype can be improved 2. Manufacturing tailor made electrodes and seamless incorporation—ideal for designing miniaturized point-of-care devices 3. Ability to design features not possible by traditional methods like rough/smooth surfaces, multiple electrodes for multiplexing, control of sensor sizes, complex geometry, cost to prototype, robustness and chemical resistance supported my availability of novel nano composite materials. Evolution of electrochemical 3D printed sensors predominantly focused on making 3D printed housing to integrate commercially available electrodes. Thus, making a functional hybrid sensor that has all the components printed and integrated will help realize the novel technologies reach the commercial arena at a scale approachable by masses. Biomarker discovery and cancer diagnostics are particularly challenged by lack of standalone devices that are sensitive to detect ultra-low levels of the biomarker levels with multiplexing capabilities. 3D printing can facilitate integration of multiple components like reagent storage and delivery, sensing surfaces by printing bio-recognition surfaces, complementary electronics for automation and data sharing modules in a miniaturized format realizing a true point of need platform.We summarized such recent hybrid systems here, Sebechlebska et al. demonstrated a 3D printed hybrid integrated sensor that integrates electrochemistry and UV/Vis absorption spectroscopy, dubbed as a first ever report of UV/Vis absorption spectroelectrochemical apparatus, employing 3D printed optically transparent working electrodes . PLA bas6 fold improvement over conventional lateral flow assays. Magnetic focus is provided by a controlling a simple magnet to manipulate magnetic probe-labelled targets with capture antibodies at the detection zones. Slower the movement the higher the interaction and higher the number of labelled probe targets at detection zone. A simple setup of 3D printed frame designed to integrate lateral flow strip with magnetic bar along with the sample application portal allowed simple operations process  for liquid biopsy and tissue samples A. The pr process B. Thorou process C.Sarioglu et al.  construcFrascella et al. demonstrated a hybrid sensor that possess active functional groups to conjugate biomolecules, they designed a photocurable formulation to introduction desired amount of carboxyl (-COOH) groups . 3D prinAbove mentioned proof-of-concept platforms highlights the need for hybrid sensors with intrinsic capabilities to evolve into true fully 3D printed diagnostic platforms. Overall, 3D printing of biosensors for diagnostics is rapidly evolving and moving in a direction to address many inherent challenges. By designing hybrid sensors with ability to integrate multiple technologies and improving biocompatibility and bio-adhering capabilities is establishing new benchmarks and a path to transform from just prototyping to commercial mass production market.Assay systems described above demonstrate that 3D printing has offered a great new low cost, asset for researchers developing biosensors. A major advantage is that sensor arrays for proteins and other analytes can be developed and fabricated on the same platform, the final optimized prototype can essentially be the final product for the clinic or hospital. That along with the speed of development and optimization, and the low cost of most of these printers, suggest a brilliant future for 3D-printed diagnostic devices measuring biomarkers of all types for cancers and other diseases. Gained protein biomarker multiplexing capabilities, increasingly supported by 3D printed immunosensors, is of great interest specially with the growing knowledge correlating different abnormalities in biomarker expression with different types of cancer. One radical foreseen change that can be realized via 3D printing, is the fabrication of sensors with integrated bio-recognition elements. Most conventional sensor assembly strategies require extensive procedures to decorate the sensor interface with biomolecules to selectively capture target analytes. This is an area where 3D printing can stand out as an approach to overcome tedious interface biomolecule decoration steps. 3D printing of biomolecules is gaining great progress in this direction specially with the rapid development in 3D bioprinting where enzymes, proteins and cells can be directly integrated in the printed matrix. Some 3D printed biosensors have been printed with integrated biomolecules  and diffIn spite of these advantages, 3D printing is still limited in aspects of multi-material printing and resolution. Developing 3D printed electrochemical sensors for cancer protein biomarkers usually necessitates multicomponent to be printed which is hindered by the very limited printing techniques capable of achieving such feature and the high variation in inter-material adhesion forces. By way of example, incorporating a conductive electrode material into a 3D printed sensor using fused deposition modeling require using conductive carbonaceous filaments that can be printed onto conventional nonconductive filaments. Although achievable , this prThese drawbacks are driving research boundaries of 3D printing to address new challenges and prior limitations. Recently, 3D printing has been utilized for in situ printing of deformable sensors right onto soft tissues and organs to accommodate its movement and expansions . This fl


Oxidative stress contributes to pathophysiological dysfunction in sickle cell anemia (SCA). Copper (Cu) is a prooxidant, whereas zinc (Zn) and selenium (Se) are antioxidant trace elements. This study investigates the serum levels of Cu, Zn, and Se among children with SCA.This cross-sectional study was performed at King Khalid University Hospital, Riyadh. Thirty-three children with SCA in steady state and 33 age- and sex-matched normal healthy children were included in the study. Cu, Zn, and Se levels were measured by inductively coupled plasma-mass spectrometry (ICP-MS) instrument.The median serum Cu levels among SCA patients (1.3 μg/mL) were higher than those of the controls . Zn (0.61 μg/mL) and Se (74 ng/mL) levels among SCA patients, however, were significantly lower than those of the controls  and , respectively. The Cu/Zn ratio among SCA patients (1.92) was higher than that of the controls (0.98).Decreased blood levels of antioxidant trace elements may contribute to the pathophysiology in SCA by promoting oxidative stress. The monitoring of trace element levels in SCA appears to be vital for decreasing morbidity associated with the disorder. Sickle cell anemia (SCA) is a hemoglobinopathy that manifests in infancy and is associated with shortened life expectancy due to repeated episodes of acute illness and organ damage [1]. It is characterized by a predominant presence of sickle hemoglobin (HbS) resulting from a substitution of glutamic acid to valine at sixth amino acid position in the β globin chain [2]. HbS is inherently unstable and tends to polymerize under low oxygen tension resulting in sickling of red blood cells, hemolysis, and anemia [3]. Sickling and hemolysis in SCA are associated with a high level of oxidative stress due to increased production of superoxide anion, reactive oxygen species (ROS), and impaired activity of ROS-scavenging enzymes [4]. Low level of ROS activity has been attributed to inadequate vitamin intake or decreased production of enzymes due to cofactors deficiency [5].Copper (Cu) and zinc (Zn) as cofactors are essential for the optimal performance of superoxide dismutase (SOD), an ROS scavenging enzyme responsible for handling superoxide radicals, thus preventing tissue damage . Zn as a trace element behaves as antioxidant [8], whereas Cu is a prooxidant [9]. SCA patients are more susceptible to alterations in these trace element concentrations because of preexisting oxidative stress due to accelerated autooxidation of HbS [10]. Selenium (Se) is essential for a wide variety of cellular functions. Human selenoprotenome comprised of 25 separate genes encodes for selenoproteins, including glutathione peroxidase, a member of ROS scavenging enzymes [11]. Se acts as an antioxidant and plays a crucial role in the defense against oxidative stress [12]. Although a number of investigations have assessed oxidative stress in SCA, the data regarding the status of anti- and prooxidant trace elements among patients with SCA are insufficient, particularly in the Kingdom of Saudi Arabia. This study was performed at King Khalid University Hospital, Riyadh, with the aim to assess serum levels of Cu, Zn, and Se among children with SCA in steady state.This cross-sectional study was performed in the Clinical Chemistry Unit at King Khalid University Hospital, Riyadh, between July 2015 and September 2016. A total of 33 patients with SCA were recruited from the hematology clinic at King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia. The diagnosis of SCA was confirmed by hemoglobin electrophoresis. Prior to enrollment in the study, it was ensured that none of the participants was receiving trace element supplements either at the time of enrollment or at least three months prior. Any patient with β- or α-thalassemia trait, G6PD deficiency, receiving treatment with hydroxyurea, receiving blood transfusion less than one month prior to enrollment, and presence of any chronic illness other than the primary disease was excluded from the study. After obtaining informed consent, 5 mL of venous blood were collected aseptically in a vacutainer without anticoagulant. Serum was separated from each sample by centrifugation and was stored at –80 °C until used. Cu, Zn, and Se levels were measured by inductively coupled plasma-mass spectrometry (ICP-MS) instrument . This study was approved by the Institutional Review Board (IRB) of the College of Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia. Reference number: 15/0292/IRB. Project number: E-15-1380. All participants/parents provided informed consent prior to inclusion in the study.Data were analyzed by MedCalc computer software, version 14.8.1. Categorical data were summarized as numbers and percentages. Numeric data were summarized as means and standard deviations, or medians and interquartile ranges. Comparison between groups for categorical markers was performed using the chi-square test or Fisher’s exact test. Comparisons between groups for continuous variables were done by using independent sample t-test or the Mann–Whitney U test. P < 0.05 was considered statistically significant. Thirty-three children with SCA  of consenting parents were included in the study. A group of 33 healthy children  was also included in the study as normal controls. Patients with SCA had low mean hemoglobin level of 91.3 ± 8.1 g/L compared to controls . The mean HbS percentage among children with SCA was 86.6 ± 7.1%, whereas none of the controls had HbS. HBA2 among children with SCA (3.4 ± 2.4%) was not different from that of the controls (2.8 ± 1.3%). Mean percentage of HBF (10.1 ± 5%) among children with SCA was higher than controls  (Table). Data comparing serum levels of Cu between patients with SCA and normal controls are shown in Figure 1. The median serum level of Cu among patients with SCA (1.3 μg/mL) was significantly higher than in normal controls . Figure 2 compares serum levels of Zn between patients with SCA and controls. The median level of Zn among the patients with SCA (0.61 μg/mL) was significantly lower than that in normal controls . Figure 3 shows the data for serum levels of Se among patients with SCA and controls. The median serum level of Se (74 ng/mL) among patients with SCA was significantly lower than that in normal controls . Cu to Zn ratio (Cu/Zn) among the control group was 0.98, whereas Cu/Zn among children with SCA was 1.92.Children with SCA had elevated serum Cu levels, whereas serum levels of Zn and Se were significantly lower than those in controls. High levels of serum Cu among SCA patients have been reported previously [13]. Free Cu ions promote the production of reactive hydroxy radicals and may therefore contribute to prooxidant activity in SCA [14]. The availability of free Cu ions is facilitated by concentrations of Cu higher than the binding capacity of ceruloplasmin serving as an antioxidant [15]. Increased urinary loses of Zn among patients with SCA [16] may also contribute to higher Cu levels and increased Cu/Zn ratio, as observed in the present study. Moreover, the higher amount of zincuria among SCA patients, by causing Zn deficiency, triggers enhanced Cu absorption from the gastrointestinal tract that may have contributed to elevated serum Cu levels [17]. Collectively, the evidence suggests that among patients with SCA, the Cu and Zn homeostasis is skewed in favor of prooxidant activity.The serum levels of Zn were significantly lower among SCA patients compared to those of controls in the present study. Elevated levels of copper and low levels of Zn among patients with SCA have been reported previously [18], indicating a compromised status of defense against oxidative stress. Zn supplementation among patients with SCA not only restores antioxidant reserves, but also decreases the incidence of infections with increased hemoglobin and hematocrit levels [19]. Restoration of serum Zn levels not only markedly reduces the tendency for sickling of red blood cells but is also associated with a remarkable reduction in the incidence of vaso-occlusive events among SCA patients . Zn deficiency has been implicated in a number of immune abnormalities [22]. Supplementation of Zn in patients with SCA has also been shown to restore impaired delayed type of hypersensitivity reaction and abnormalities in natural killer cell function [19]. The diversity of abnormalities described among SCA patients [22] may be linked directly or indirectly with Zn deficiency, as Zn has been shown to be an essential cofactor for over 300 enzymes [23].SCA patients in the present study had a higher Cu/Zn ratio compared to normal controls. Increased Cu/Zn ratio has been documented in SCA previously and is considered a useful adjunct for the diagnosis of SCA [24]. Successful treatment of hyperglycemia with resveratrol in type 2 diabetes mellitus is associated with a remarkable reduction in oxidative stress along with decreased Cu/Zn ratio due to the lowering of serum Cu levels [25]. These findings indicate that the assessment of Cu/Zn ratio may serve as a useful tool for gauging oxidative stress. Periodic assessment of Cu/Zn ratio among SCA patients, therefore, appears to be useful not only for monitoring oxidative stress among these patients, but may also preempt an impending sickle cell crisis.Compared to normal controls, serum levels of Se were significantly lower among SCA patients in the present study. Though infrequently, low blood levels of Se have been reported among patients with SCA [26], suggesting a weak antioxidant potential. The antiinflammatory role of Se was recently demonstrated in an experimental study on adiponectin knockout mice where Se was shown to provide protection against chronic inflammation-induced colon cancer [27]. Selenoproteins are Se containing proteins mediating biological effects of Se [28]. Glutathione peroxidases are selenoproteins and are the main antioxidants involved in neutralizing the production of ROS [29]. Low levels of glutathione peroxidase among patients with SCA [26] highlights the importance of Se in handling the oxidative stress associated with SCA. In addition, Se has been shown to provide protection against oxidative stress-induced lipid peroxidation and disturbances in fatty acid biosynthesis among patients with SCA [30].In conclusion, children with SCA had low levels of Zn and Se with elevated serum levels of Cu. Decreased blood levels of antioxidant trace elements may contribute to the pathophysiology in SCA by promoting oxidative stress. The monitoring of trace element levels in SCA appears to be important for reduction in disease associated morbidity among children with SCA. Large-scale studies are recommended to validate the findings of this study and further elucidate the role of trace elements in sickle cell disease.The author would like to thank Mr. Mohammed Atiya for his assistance with the experimental work.