Klotho is an antiaging protein which exerts known cardioprotection. In kidney, trans-membrane Klotho acts as essential co-receptor of fibroblast growth factor 23 (FGF23). In the heart, soluble Klotho (sKlotho) protects from systolic dysfunction independently of FGF23. Here, we analyzed the association of FGF23 and sKlotho upon progression of chronic heart failure (CHF) and analyzed Klotho expression in human hearts. Serum levels of sKlotho and FGF23 were measured in 287 patients with cardiomyopathy (CMP). Tissue samples from CMP (n = 10) and healthy control hearts (n = 10) were analyzed for Klotho mRNA and protein expression. Individuals in the first FGF23 tertile were 4.1 times more likely of freedom from death, heart transplantation or assist device implantation compared to third tertile. No relationship was found between sKlotho and the combined endpoint. Instead, Klotho mRNA encoding the full-length form was upregulated in human CMP hearts. Immunoblotting confirmed upregulation of sKlotho associated with increased expression of proteases involved in cleavage of Klotho suggesting rather local effects of Klotho in the heart. Therefore, we conclude that in contrast to FGF23, serum sKlotho is not associated with disease severity or progression in CHF. Instead, Klotho is expressed and upregulated in diseased hearts, suggesting local paracrine effects. Hence, strong emphasis is put on the identification of new targets to better understand the complex pathophysiology of CHF and develop novel treatment strategies.Chronic heart failure (CHF) remains a major public health issue. Prevalence of CHF is constantly rising and despite the best current treatment, disease-related hospitalizations are frequent and outcome is poor, with a one-year mortality rate of 6% to 24%8. The classical effects of FGF23 in the kidney and parathyroid glands are mediated by its binding to FGF receptor-1 (FGFR-1) complexed to the co-receptor Klotho, thereby increasing the binding affinity of FGF23 for FGFR10. Activation of the membranous Klotho-FGFR co-receptor complex by FGF23 suppresses renal synthesis of 1,25 dihydroxyvitamin D and inhibits renal phosphate reabsorption12. In the heart FGF23 has direct hypertrophic effects on cardiomyocytes mediated via FGF receptor-dependent activation of the calcineurin-NFAT signaling pathway5. Klotho is a type 1 membrane protein that exerts known antiaging function and is actively involved in the prevention of arteriosclerosis15. Deficiency of Klotho is known to shorten lifespan and leads to a phenotype resembling human aging13, whereas overexpression of Klotho extends lifespan by 20–30%15. Klotho is predominantly produced in the kidney, and circulating levels of soluble Klotho (sKlotho) are reportedly decreased in chronic kidney disease (CKD)17. The extracellular domain of Klotho can be cleaved by the α-secretases ADAM10 and ADAM17 and released into the circulation18. A first cut close to the cell membrane  results in a 130 kDa circulating protein, and a second cut (beta-cut) between the KL1 and KL2 domains produces two smaller proteins with molecular sizes of approximately 65 kDa19. Both the full-length and the shorter forms of soluble Klotho (sKlotho) are hormones, with distinct but overlapping effects. In addition, a secreted isoform (isoform 2) arising from alternative RNA splicing has been described, however, does not seem to be translated into a functional protein20. In rodents sKlotho exerts cardioprotection through downregulation of TRPC6 calcium channels21. Moreover, in a CKD mouse model decreased levels of circulating sKlotho were an important cause of uremic cardiomyopathy independently of FGF2322. In hemodialysis patients, higher sKlotho levels were associated with the absence of atrial fibrillation23. From these data it appears that sKlotho might oppose the detrimental effects of FGF23 on the heart. Interestingly, so far Klotho has not been detected in cardiomyocytes13.In clinical studies elevated levels of phosphatonin fibroblast growth factor 23 (FGF23), an endocrine hormone secreted from osteoblasts and osteocytes that regulates phosphate homeostasis, have been linked to cardiac remodeling and the advent and progression of CHFBased on the data published on Klotho in cardiac remodeling, we hypothesized that sKlotho might be inversely related with disease severity and progression in CHF. Furthermore, we examined the role of Klotho in human specimens of cardiomyopathy (CMP) and non-failing hearts.The study cohort of 287 patients with chronic heart failure due to non-ischemic CMP and reduced left ventricular ejection fraction (LV-EF) comprised idiopathic CMP (47%), virus-negative inflammatory CMP (33%), and various etiologies including hypertrophic CMP, cardiac amyloidosis, and cardiac sarcoidosis (20%).Median levels of FGF23 and sKlotho in the plasma were 21.8 RU/ml (IQR 12.1–45.7) and 380 pg/ml (IQR 301–529), respectively. Baseline characteristics according to the median of FGF23 are shown in Table Complete follow-up information was available in 283 (98.6%) patients. Thirty-seven (13.1%) patients reached the combined endpoint of death, heart transplantation or assist device implantation  within a median follow-up period of 634 [296–1073] days. In these patients’ serum levels of FGF23  were higher as opposed to lower serum levels of sKlotho .In Cox regression analysis adjusted for age and sex, FGF23 , but not sKlotho , was associated with an increased risk for the combined end point of death, heart transplantation or assist device implantation. Moreover, in tertile-based sex-adjusted analysis, individuals in the first FGF23 tertile were 4.1 times  more likely of freedom from the combined end point compared to individuals in the third tertile Fig. . No relaSince we found no clear relation of sKlotho in the serum to heart failure outcomes we aimed to investigate Klotho expression directly in the human heart. 3′ RACE PCR utilizing oligo dT primers followed by RT-PCR analysis with isoform specific primers revealed expression of full-length Klotho mRNA in human heart and kidney Fig. . In contNext, we examined Klotho protein expression in heart tissue derived from CMP and control hearts by Western blot analysis. Tissue samples from kidneys were used as positive controls. As shown in Fig. Since ADAM10, ADAM17 and BACE1 are known proteases involved in the cleavage of Klotho, we analyzed the expression of these enzymes in human heart and kidney. As shown in Fig. In this longitudinal study we demonstrate that FGF23, rather than sKlotho in the serum, appropriately reflects disease severity and progression in CHF. Instead, we show for the first time Klotho mRNA and protein upregulation in human cardiomyopathy, independent of sKlotho levels in the serum suggesting paracrine local affects.25. Also, we show a clear dose-response relationship for FGF23 levels with increasing severity of HF. Previous findings from population-based studies demonstrated an association between FGF23 and the advent of incident HF27. In addition, circulating FGF23 concentrations have been linked to left ventricular hypertrophy (LVH)28. Corresponding results by Faul et al. demonstrated an independent association between elevated FGF23 levels and LVH in a large CKD cohort5. Data from the same group indicate hypertrophic growth in isolated neonatal rat cardiomyocytes mediated by FGFR-dependent activation of the calcineurin-NFAT signaling cascade, not requiring Klotho as coreceptor29.The present analysis confirms the association between FGF23 and disease progression in HF and its potential as a biomarker for risk prediction22. Thereby, sKlotho interrupts a feed-forward loop that amplifies stress-induced abnormal intracellular Ca2+ signaling, which activates the calcineurin-NFAT signaling cascade. In CKD mice Klotho deficiency correlates with the extent of cardiac hypertrophy and fibrosis30. Also, in rodents sKlotho protects against uremic cardiomyopathy independently of FGF23 and phosphate21. Findings from a clinical study have linked higher Klotho levels to the absence of atrial fibrillation in hemodialysis patients23.While Klotho has been studied extensively in experimental models and in patients with CKD, data in human HF are rare. Experimental data in mice suggest that sKlotho exerts cardioprotection through downregulation of the TRPC6 calcium channel31. This is in line with findings in patients with renal disease showing significantly lower sKlotho levels than in healthy controls23 and decreased Klotho expression early in the course of CKD33. This phenomenon was explained by the fact that sKlotho derives mainly from renal tubular cells9. Findings in our cohort with stable HF do not support a protective effect of circulating sKlotho in heart failure. Although absolute differences in sKlotho levels between patient groups with and without an endpoint were small albeit significant, sKlotho levels were not associated with event-free survival. Corresponding results were recently reported in patients with CKD and ESRD33.In contrast to FGF23, we could not find a clear relationship between sKlotho in the serum and HF severity. Nevertheless, sKlotho levels in our HF cohort were lower by trend than recently reported by a healthy Danish cohort using the same ELISA kit (380 pg/ml vs. 472 pg/ml)13. Accordingly, we also failed to detect expression of Klotho in mouse hearts indicating species-specific differences. Using different primer pairs and sequencing of 3´RACE generated cDNA products clearly showed that Klotho mRNA indeed is expressed in the human heart. Klotho amplification in human hearts may have been overlooked in earlier studies since expression of mRNA occurred only after 26 PCR cycles and other primers were used. Based on our experiments we conclude that full-length but not the secreted from of Klotho is expressed in the human heart. Our results were further confirmed by immunoblotting, and immunohistochemistry. Immunoblotting with two different Abs recognizing binding sites in the KL1 region of Klotho revealed a single 65 kDa band corresponding most likely to the cleaved form of sKlotho in the heart34. The assumption that upregulated Klotho protein in diseased hearts is derived from cleavage of the membrane-bound Klotho is further supported by our findings that (i) circulating sKlotho levels in serum were neither upregulated nor associated with disease progression in CHF and that (ii) full-length Klotho mRNA was upregulated in diseased hearts, whereas mRNA of secreted Klotho (isoform 2) was not detectable. Furthermore, a recently published study showed that the secreted form of Klotho mRNA (isoform 2) contains premature termination codons priming mRNAs for degradation by nonsense-mediated mRNA decay thereby abolishing protein translation35. Moreover, we detected the expression of proteases like ADAM10, ADAM17 and BACE1 capable for cleavage of Klotho at the transmembrane site  and between the domains KL1 and KL2 (beta-cut)34. The fact that we could only detect the 65kDA protein, suggests complete cleavage of transmembrane Klotho into KL1 and KL2 domains in the heart by proteolysis. Interestingly, production of Klotho is 200fold higher in the kidney, but expression of proteolytic enzymes is on the same level as compared to heart tissue. Incomplete cleavage might explain the presence of two bands (130 kDa and 65 kDa) in the kidney compared to the heart. Therefore, our results support the hypothesis that Klotho expression in the heart is independently regulated to serum levels and might be controlled in a local paracrine manner.Instead, we provide for the first-time unambiguous evidence for Klotho mRNA expression in human hearts. Particularly the full-length transcript of Klotho was significantly upregulated in CMP hearts compared to non-failing controls. In contrast to the kidney, we could only detect the full-length trans-membrane form of Klotho mRNA in the heart, whereas the secreted form (isoform 2) was not detectable. Previous reports indicating no relevant expression of Klotho in cardiomyocytes might be explained by the fact that Klotho expression was studied mainly in mice and rodent cell lines, whereas data regarding Klotho expression in the human heart is very limited37. Low circulating Klotho levels in HF and high expression of Klotho in cardiomyopathy, however, are difficult to reconcile. In rodents sKlotho ameliorates cardiac hypertrophy by inhibiting TRPC6 currents in cardiomyocytes21. This mechanism is mediated by the insulin-like growth factor 1 receptor and is independent of FGFR and the Klotho receptor. Furthermore, sKlotho protects from isoproterenol induced cardiomyocyte apoptosis via inhibition of p38 and c-Jun NH2-terminal kinase pathways suggesting ameliorating stress induced cardiac damage38. A recently published study suggests that sialogangliosides in lipid rafts can act as membrane receptors for sKlotho and that particularly the KL1 domain is sufficient to inhibit raft-dependent PI3K signaling and TRPC6 function thereby protecting against stress-induced cardiac hypertrophy in mice39. In fact, the antibodies used in our experiments mainly recognize the KL1 domain (65 kDa) of sKlotho. From this we hypothesize that local overexpression of sKlotho may compensate for Klotho-independent maladaptive effects of FGF23 in an autocrine or paracrine manner in human heart failure.Elevated levels of circulating FGF23 are in line with previous findings demonstrating its role in cardiac remodeling and disease progression. Our results support the notion that FGF23 is upregulated in diseased human CMP hearts and might contribute to increased serum levels in chronic heart failure even prior to detoriation of renal functionData in this study are derived from a single-center cohort of patients with non-ischemic cardiomyopathy. Hence, applicability of findings to patients with ischemic cardiomyopathy is limited. Owing to its nature as a cohort study, our trial lacks a control group that would allow for unequivocal comparison of sKlotho levels. Furthermore, the trigger of local Klotho overexpression remains unresolved and needs to be clarified in further experimental models.In this study we show that FGF23 rather than sKlotho in the serum is associated with disease severity and progression of chronic heart failure. Instead, Klotho is upregulated locally in CMP hearts, suggesting paracrine effects.An expanded Methods section on real-time RT-PCR, western blotting, and immunoperoxidase staining is provided in the Supplementary Material. A complete list of amplification primers used in the study is provided in Supplementary Table For this retrospective analysis 287 Caucasian patients with chronic heart failure due to non-ischemic cardiomyopathy and reduced LV-EF, who were referred for further invasive diagnostic evaluation between January 2007 and September 2014 were consecutively included. Inclusion criteria were patients ≥18 years of age with confirmed diagnosis of CHF based on the presence of current or previous symptoms or characteristic clinical signs and evidence of left ventricular dysfunction and normal or mild reduced kidney function. Exclusion criteria were patients with acute HF, vitamin D and/or calcimimetic therapy for the last six months, coronary artery disease (CAD) on coronary angiography or moderate-severe CKD to obviate potential interferences. Patients were treated according to prevailing CHF guidelines. Patients were followed until December 2014 (time of data censoring) or to the occurrence of death, heart transplantation or ventricular assist device (VAD) implantation, which constituted the combined endpoint. Death events were retrieved from the local mortality registry and from the patients’ relatives.Ventricular tissue samples were either obtained by endomyocardial biopsy (EMB) or as dispensable tissue during heart surgery from patients with non-ischemic cardiomyopathy (n = 10) or healthy donor hearts (n = 10) before transplantation. Samples were immediately immersed in RNAlater (Qiagen GmbH) and stored at 4 °C for 24 hours (RNA later incubation time) followed by long-term storage at −80 °C for further RNA processing. Samples were analyzed for the expression of Klotho and BNP mRNA. Healthy kidney tissue (n = 4) was obtained from non-diseased parts of tumor nephrectomy samples. Mouse wild type (WT) and Klotho knock-out (KO) tissues from kidney and heart were kindly provided by Dr. Leifheit-Nestler lab  and Dr. Jakob Völkl .The study confirmed with the principles outlined in the Declaration of Helsinki and was approved by the local ethics committee of the Medical University of Innsbruck. All patients gave written informed consent for participation in the study.Fasting blood samples were drawn in all patients at study entry and stored at −80°. All laboratory variables were measured at the central laboratory of Innsbruck Medical University Hospital that undergoes regular internal and external quality audits. FGF23 was determined using an FGF23 immunoassay  with polyclonal antibodies detecting both the intact molecule and large carboxyl terminal fragments of human FGF-23. Circulating concentrations of FGF23 are expressed as relative units per milliliter (RU/ml).Soluble α-Klotho was measured with a solid-phase sandwich ELISA . This assay detects the tertiary protein structure of an extracellular domain of KL1 with monoclonal antibodies. Circulating concentrations of soluble α-Klotho are expressed as pg/ml.Freshly isolated tissues from human CMP, non-failing hearts and kidneys were immediately fixed in 10% neutral buffered formalin for further processing. An expanded Methods section on real-time RT-PCR, western blotting, and immunoperoxidase staining is provided in the supplement.Data are presented as median  or n (%), as appropriate. The Shapiro-Wilks test was used to test for normality in distribution of included parameters. We used the Kruskal-Wallis and Pearson Chi-square tests or one-way ANOVA to test for the differences in categorical or continuous factors, respectively, between various categories of FGF23 and sKlotho. Spearman rank correlation was used to assess cross-sectional relations between FGF23, sKlotho and 25 hydroxyvitamin D (25(OH)D). Assessment of the prognostic relevance of FGF23 and sKlotho per standard deviation each for the combined endpoint of freedom from death, heart transplantation or assist device implantation was performed using sex- and age-adjusted Cox proportional hazards regression analysis. Kaplan-Meier estimates and the log-rank test were used to calculate freedom from death, heart transplantation or assist device implantation. A multivariate model for FGF23 and sKlotho per log unit each was calculated with eGFR, NT-proBNP, and LV-EF as co-variates. Differences in mRNA expression by quantitative PCR between biopsies obtained from kidney, diseased hearts and healthy controls were analyzed by one-way ANOVA for multiple group comparisons. P values < 0.05 were considered to indicate statistical significance. Statistical analysis was performed using SPSS 20.0 (SPSS Corp.).All data generated or analysed during this study are included in this published article and its Supplementary Information files.Supplementary Material


Perspectives” article, we review and comment on current advances in Shank3 research and include some original data that show common Shank3 deficits in a number of seemingly unrelated human neurological disorders that include sporadic Alzheimer’s disease (AD), autism spectrum disorder (ASD), bipolar disorder (BD), Phelan–McDermid syndrome , and schizophrenia (SZ). Shank3 was also found to be downregulated in the CNS of the transgenic AD (TgAD) 5x familial Alzheimer’s disease murine model engineered to overexpress the 42 amino acid amyloid-beta (Aβ42) peptide. Interestingly, the application of known pro-inflammatory stressors, such as the Aβ42 peptide and the metal-neurotoxin aluminum sulfate, to human neuronal–glial cells in primary culture resulted in a significant decrease in the expression of Shank3. These data indicate that deficits in Shank3-expression may be one common denominator linking a wide-range of human neurological disorders that exhibit a progressive or developmental synaptic disorganization that is temporally associated with cognitive decline.Signaling between neurons in the human central nervous system (CNS) is accomplished through a highly interconnected network of presynaptic and postsynaptic elements essential in the conveyance of electrical and neurochemical information. One recently characterized core postsynaptic element essential to the efficient operation of this complex network is a relatively abundant ~184.7 kDa proline-rich synapse-associated cytoskeletal protein known as Shank3 . In this “ SH3 and multiple ankyrin repeat domain” proteins known as Shank1, Shank2, and Shank3 encode abundant postsynaptic scaffolding proteins highly enriched at glutamatergic synapses in the human and murine central nervous system (CNS)  . All threm (CNS) , 6. As pem (CNS) , 7. To aem (CNS) , 9. Diffem (CNS) ; (iii) pem (CNS) –11; and em (CNS) –17. Thisem (CNS) , 18, 19.loss-of-function” mutations or altered Shank3 expression can lead to such phenotypic diversity.As forementioned alternate Shank3 protein isoforms are differentially expressed according to developmental stage, cell type, and course of aging, suggesting the existence of isoform-specific roles for Shank3 at varying subcellular localizations, along different CNS neurites and synaptic endings at different stages of development and disease. Shank3 mutations that include gene breakpoints, deletions, point mutations, missense mutations, microdeletions, and nonsense mutations are associated with moderate to severe intellectual disabilities –7. AD wiThe 42 amino acid amyloid-beta peptide and aluminum (sulfate) were chosen as highly relevant pathogenic neurological stressors for the experimental treatment of HNG cells and assessment of Shank3 expression for the following reasons  altered neurogenesis and disrupted expression of synaptic proteins that affect synaptic organization and plasticity; (ii) altered activation of cell adhesion molecules such as integrin; (iii) developmental and morphogenetic abnormalities; (iv) changes in biometal abundances and transporters such as those for zinc; (v) alterations in G protein-coupled metabotropic glutamate receptor 5 (mGluR5)-Homer scaffolds and mGlu5 receptors; and (vi) peripheral aberrations such as abnormally heightened sensitivity to pain in diseases associated with Shank3 deficits or in Shank3 animal models  propose that pharmacological approaches including anti-microRNA strategies directed toward the manipulation of Shank3 and/or related synaptic protein expression may be useful in the clinical management of multiple neuropsychiatric disorders, which exhibit an underlying commonality in intellectual disability and/or progressive cognitive decline.Importantly, these experimental interpretations should remain be somewhat speculative as they are derived from preliminary studies, and more research is required to further clarify the significance of Shank3 deficits in other neurological disorders and experimental models. Whether Shank3 downregulation is a direct cause or consequence of these neurological disorders, or an unrelated epiphenomenon remains open to question. However, several recent studies of Shank3’s extensive and remarkably diverse interactome further underscore the idea that this highly interconnected synaptic protein is a “l models , 51–61. All procedures involving murine and postmortem human tissues were followed and handled in strict accordance with the ethics review board policies at donor institutions and the Institutional Biosafety Committee/Institutional Review Board (IBC/IRB) ethical guidelines at the LSU Health Sciences Center, LA 70112 .PA, LC, VJ, and YZ performed the experiments; YZ and WL analyzed the data; WL organized the data and wrote the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PON1) that persisted in early childhood and was attenuated in mid-childhood blood. Cord blood methylation at the PON1 locus predicted lower cognitive test scores measured during early childhood. Methylation at the PON1 locus was associated with PON1 expression in an independent set of cord blood samples. The observed persistent epigenetic disruption of the PON1 gene may modulate mercury toxicity in humans and might serve as a biomarker of exposure and disease susceptibility.Prenatal exposure to mercury, a known neurotoxic metal, is associated with lower cognitive performance during childhood. Disruption of fetal epigenetic programming could explain mercury’s neurodevelopmental effects. We screened for epigenome-wide methylation differences associated with maternal prenatal blood mercury levels in 321 cord blood DNA samples and examined the persistence of these alterations during early  and mid-childhood . Among males, prenatal mercury levels were associated with lower regional cord blood DNA methylation at the Paraoxonase 1 gene ( Since the industrial revolution, mercury levels in surface water have tripled2. For the general public, the consumption of fish or seafood containing MeHg is the major source of exposure3.Mercury (Hg) is a ubiquitous worldwide environmental contaminant that can persist in the environment and bioaccumulates as methylmercury (MeHg) in the food chain. Elemental mercury is a natural metal that is primarily introduced in the air, water and food from coal burning power plants, artisanal mining activities and industrial applications4. Prenatal effects of acute MeHg exposure were first documented in 1956, when industrial activity heavily polluted Japan’s Minamata bay causing coastal residents to consume highly contaminated seafood. Some infants born during this period suffered from severe neurological disabilities and fetal abnormalities5. Recent prospective epidemiological studies have shown that even moderate or low-level prenatal exposure to mercury in utero typical of regular fish consumption may be associated with lower cognitive test scores in children10. However, the dose-response relationship, mechanism of action and potential effect modifiers such as gender and timing of exposure have not been fully evaluated11.MeHg crosses the placenta and the blood-brain barrier and accumulates in fetal tissues resulting in fetal blood concentrations that typically exceed maternal levels12. The ability of mercury to cross the placenta and blood-brain barrier during development makes it a candidate toxicant for the disruption of fetal programming events that could propagate through different germ layers during embryogenesis.The specific mechanisms of neurotoxicity and cognitive disruption associated with prenatal mercury exposure remain poorly characterized in humans. Nonetheless, the period of fetal development has been shown to be sensitive to prenatal exposure, perhaps in part because of the dramatic DNA methylation changes and epigenomic remodeling that takes place early during embryogenesis, giving rise to cells and tissues with specific DNA methylation patterns13. Evaluating the persistence of epigenetic modifications is critical as DNA methylation is a dynamic process that can drift with age and other stochastic processes14. To date, three epigenome-wide association studies have been conducted identifying unique genomic regions as well as individual CpG methylation disruption in cord blood16 and placenta17 of newborns prenatally exposed to mercury. However, these studies were limited in sample size (ranging from 41 to 141 samples) and did not evaluate whether the observed epigenetic alterations were persistent, sex-specific or related to cognitive performance in childhood. Epigenetic responses to environmental conditions can be sex specific in terms of perception of exposure and subsequent response, particularly for neurodevelopmental disorders19. Furthermore, gender differences in MeHg neurotoxicity have been documented in epidemiological and animal models21. Therefore, it is crucial to evaluate sex-specific epigenetic alterations in response to environmental conditions.Although several studies have characterized DNA methylation changes in cord blood relative to prenatal environmental exposures, very few studies have evaluated the persistence of these changes into childhoodIn the present study, we examined associations of prenatal mercury exposure with epigenome wide DNA methylation in cord blood, and evaluated if these epigenetic modifications persist in blood through early and mid-childhood. We also evaluated sex-specific differences in response to prenatal mercury exposure. We hypothesized that persistent DNA methylation modifications in cord blood related to prenatal mercury exposure would also be associated with cognitive test scores measured in early childhood.2-transformed it for analyses to approximate a log-normal distribution, Supplementary Fig. A total of 321 mother-child pairs had available data on both maternal prenatal mercury exposure and cord blood DNA methylation. During early childhood (range: 2.9 to 4.9 years), 75 participants had whole blood samples available for analysis and 291 participants had samples available for mid-childhood analyses (range: 6.7 to 10.5 years), Table PON1) was hypomethylated by prenatal mercury exposure among boys , Fig. PON1 DMR ranged from a 1% to a 3.8% decrease in individual cord blood CpG methylation per doubling in maternal RBC-Hg concentrations, Table PON1 locus were inversely associated with maternal RBC-Hg concentrations , Table PON1 DMR are shown in Fig. To identify Differentially Methylated Regions (DMRs) in the genome relative to prenatal mercury exposure, we implemented a regional based approach that is agnostic to both genomic annotation and direction of individual CpG associations. After stratifying on sex, a DMR covering 9 CpGs of chromosome 7 in the Paraoxonase 1 gene , a doubling in prenatal mercury concentration was associated with a 4.6% decrease in mean methylation levels of the DMR in PON1, measured across the nine CpGs . Investigating all 9 CpGs individually, the inverse adjusted association of prenatal mercury exposure reached statistical significance in early childhood among five CpGs: cg07404485 , cg05342682 , cg21856205 , cg01874867  and cg20119798 . In mid-childhood (n = 149), mean methylation levels of the PON1 DMR were marginally associated with prenatal mercury exposure for males . However, five individual CpG sites within the PON1 region were associated with maternal RBC-Hg concentrations: cg05342682 , cg19678392 , cg21856205 , cg17330251  and cg01874867 . Persistence of the association for the male-specific DMR in PON1 and prenatal RBC-Hg concentrations is summarized in Table To evaluate the persistence of association between prenatal mercury exposure and DNA methylation at the WBP11P1) located in chromosome 18 . However, this association did not persist in early  or mid-childhood , Table In adjusted robust linear regression models, one CpG was differentially methylated relative to prenatal mercury exposure after using a Bonferroni adjusted level of significance, Fig. TOR4A) was hypermethylated relative to maternal RBC-Hg concentrations , Fig. P = 0.34) but not significant, likely due to sample size (N = 37). However, the association for this CpG was significant for methylation levels measured in blood at mid-childhood , Table After stratifying by sex, one CpG in males cg13416866) annotated to the Torsin Family 4, Member A gene 22. In this independent set of samples we observed a moderate negative correlation between PON1 RNA expression and mean methylation levels of the nine CpGs in the DMR that approached statistical significance , Fig. PON1 DMR located in the body of the gene and in the north shore region of a CpG island had the strongest association with expression: cg07404485  and cg05342682 , respectively. Five other loci from the DMR reached marginal significance (P < 0.10), Fig. PON1 and annotation for the gene expression array used, mRNA, known SNPs and CpGs are shown in Supplementary Fig. To evaluate the functional relevance of methylation changes, we examined whether DNA methylation of the PON1 DMR were marginally associated with cognitive PPVT scores measured in early childhood . Within the PON1 DMR, four individual CpGs were significantly associated with cognitive scores measured in early childhood among males. Specifically, an inverse multivariate adjusted association was observed between DNA methylation levels and the PPVT cognitive scores for cg05342682 , cg21856205 , cg17330251  and cg01874867 . No significant associations were observed for individual CpG methylation levels of this region and WRAVMA cognitive test scores in males, Table PON1 methylation in females, mean cord blood methylation levels of the PON1 DMR was associated with lower cognitive test scores for the PPVT in girls measured in early childhood  but not WRAVMA scores, Supplementary Table WBP11P1 gene were marginally associated with WRAVMA cognitive test scores in the entire sample , Supplementary Table 8, we did not observe evidence for an association between log2-tranformed RBC-Hg and PPVT cognitive scores  or with WRAVMA scores  in our smaller sample. Therefore, we were unable to estimate the mediated effects of exposure on cognitive performance in this subsample of the cohort.In males, mean methylation levels in cord blood for the PON1 DMR measured in cord blood and in early or mid-childhood , Fig. Finally, we observed similar DNA methylation levels at the ood Fig. . Among 2PON1 gene relative to prenatal mercury exposure in umbilical cord blood from males but not females, an association that persisted during early and mid-childhood. We also showed that higher DNA methylation levels of the PON1 region are associated with lower cognitive test scores in early childhood for both sexes. Based on publicly available reference datasets we observed that cord blood DNA methylation levels of the PON1 region is associated with decreased PON1 expression in cord blood. In individual CpG analyses, only one CpG of the WBP11P1 gene was associated with prenatal mercury exposure, but this association did not persist in early or mid-childhood. In male-specific site-by-site analyses, a CpG of the TOR4A gene was hypermethylated relative to prenatal mercury exposure and this association persisted in mid-childhood. Taken together, these results suggest that moderate mercury exposure during pregnancy can lead to sex-specific functional epigenetic alterations that persist throughout childhood and are associated with cognitive performance.In this study we observed DNA methylation changes of a genomic region of the PON1) belongs to the serum paraoxonase family of enzymes previously shown to provide oxidative protection for high density lipoproteins and hypothesized to guard against atherosclerosis and metabolic syndrome24. PON1 is synthesized in the liver and secreted into the plasma where it can metabolize toxic oxidized lipids25. The main substrates of PON1 are lactones, but promiscuous catalytic activity towards organophosphate triesters, arylesters, cyclic carbamates, glucuronides, oestrogen esters and thiolactones has also been documented26. Genetic polymorphisms in PON1 and enzyme activity have been associated with adverse perinatal health outcomes such as decreased fetal growth, pre-term birth and reductions in head circumference28. Genetic variants as well as low enzymatic activity of PON1 have been shown to contribute to the neurodevelopmental toxicity of organophosphorus (OP) pesticides in animal models and human studies30. PON1 protein has also been localized in brain regions of animal models as well as human tissue and experimentally shown to interact with other proteins in the brain that are essential for brain homeostasis31.The Paraoxonase 1 or arylesterase 1 gene (In vitro models and human studies have shown that MeHg exposure exerts an inhibitory effect on PON1 enzyme activity even after controlling for genetic variants including SNP-108 (rs705379), one of the major genetic determinants of PON1 expression34. The inhibitory mechanism of MeHg on PON1 has been hypothesized to be involved in the development of cardiovascular disease, potentially explaining epidemiological associations with MeHg35. Furthermore, PON1 enzyme activity is lower in neonates but relatively stable in adults exhibiting age-dependent developmental changes through at least 7 years of age. The developmental change in enzyme activity might create a sensitive window for toxic exposures during fetal development37. In fact, hypersensitivity to mercury exposure  among infants exposed to mercury has been attributed to genetic variation and subsequent enzyme activity of PON138. A large prospective epidemiological study showed that PON1 genotype can modify the association of prenatal MeHg exposure and neurodevelopment, leading to stronger adverse effects in IQ deficits during childhood39. Interestingly, in a study of 896 Inuit adults, blood MeHg concentrations lowered PON1 enzyme activity independent of genetic variation, and there was no evidence of an interaction between MeHg and genetic variants of PON132. The lack of gene-environment interaction suggests a third control mechanism involved in the relationship between genetic variation, exposure and subsequent enzyme levels.PON1 genetic variants are better characterized, less is known about epigenetic control mechanisms. In a recent integrative genomic study, a single nucleotide polymorphism in PON1  was shown to be strongly associated with DNA methylation subsequently mediating PON1 enzyme activity in newborns, an association that persisted to nine years of age40. In another study of obese adults, a similar inverse correlation between PON1 methylation and catalytic activity of the enzyme was documented41. This is consistent with our observation in which increasing methylation levels at the PON1 DMR decreased gene expression using data from an independent set of cord blood samples. These results highlight DNA methylation as a control mechanism involved in PON1 expression, underscoring the importance of the male-specific PON1 hypomethylation observed in our study relative to prenatal mercury exposure and subsequent association with cognitive test scores.Although PON1 are unknown for humans. However, in a case-control study of myocardial infarction, female specific hypermethylation of PON1 was observed among cases compared to controls42. Moreover, animal studies have documented male-specific histone modifications along with increased expression of PON1 in male offspring exposed to a high fat diet in utero43. This is consistent with our data in which male-specific hypomethylation of PON1 was observed for prenatal mercury exposure, potentially leading to an increase in expression. The observed sex-specific epigenetic modification might help explain sex differences observed in mercury’s toxicity. For example, male-specific mercury associations have been documented in epidemiological studies for metabolic syndrome44, ADHD-related behavior, and neurodevelopment46. An experimental study of mice prenatally exposed to mercury demonstrated that males have a higher expression of immune-related genes and miRNA levels compared to females, suggestive of potential sex-specific response during fetal development47.Sex-specific differences in DNA methylation for PON1 region could serve as a mediator of genotype and gene expression, explaining age-dependent changes in enzyme activity and modulation of mercury’s neurotoxicity, as shown for OP pesticide exposure. However, our study lacks information on genetic variants to estimate the effect of DNA methylation conditional on genotype. Therefore sex-specific epigenetic differences in the region could partially be driven by genetic variation. Nevertheless, increasing levels of DNA methylation in the PON1 region was associated with a modest decrease in PPVT cognitive scores in both males and females, suggesting that epigenetic control of PON1 could be implicated in cognitive development independent of prenatal mercury exposure or sex. As supported by the observed methylation-expression relationship in the BEAR birth cohort22 and previous studies40, DNA methylation regulates expression and enzymatic activity of PON1 providing a potential mechanism of mercury’s associated neurotoxicity. Furthermore PON1 might also serve as an important target for other toxic effects of mercury exposure.DNA methylation of the PON1 has been so far focused in cardio-metabolic outcomes, there is evidence that suggests that PON1 genotype and enzyme activity is associated with adult neurological diseases such as Parkinson and Alzheimer48. Furthermore, PON1 genetic variants and enzymatic activity have been linked to autism spectrum disorder in cross-sectional studies50. However, we did not measure DNA methylation in the brain as a target tissue a common limitation of prospective epidemiological studies.Although the growing interest in WBP11P1 gene (cg13340705). The WW domain binding protein 11 pseudogene 1 (WBP11P1) has no known function and the association was not persistent in early or mid-childhood suggesting, that this modification is transient at birth. The single CpG (cg13416866) associated with prenatal mercury exposure in males in the Torsin Family 4, Member A gene (TOR4A) persisted into mid childhood but was not associated with cognitive scores. However, these individual loci have not been previously implicated in mercury toxicity, and the evidence for functional or phenotypic association with cognitive performance was limited.The CpG found to be differentially methylated for the entire sample relative to prenatal mercury exposure is located in the TSS1500 region of the EMID2 gene was associated with prenatal mercury exposure and neonate behavior. Interestingly both the EMID2 and PON1 gene are located in a region of chromosome 7 (q21–q31), experimentally shown to be sensitive to genomic imprinting using human-mouse monochromosomal hybrids51. Additionally, this region in PON1 has been shown to have parent of origin specific methylation involved in the etiology of Silver-Russell syndrome, but PON1 remains a disputed imprinted gene in humans52. Future studies should evaluate if prenatal mercury exposure targets other imprinted genes from this region. Heterogeneity of the DNA methylation targets across studies suggests that mercury’s associated epigenetic disruption might be time-sensitive during fetal development, emphasizing the importance of collecting both sensitive and time-specific biomarkers of exposure. Furthermore, mercury exposure levels in our cohort were relatively low but comparable to the general US population53. Therefore our results might not be generalizable to populations exposed to higher levels of mercury.Finally, in the three previous epigenome-wide studies of prenatal mercury exposure no overlaps between differentially methylated regions or CpGs have been documented. One study that included epigenome-wide analysis of placenta samples found that DNA methylation of the Our study has several limitations. First, in the absence of genotype information it is not possible to determine if genetic variants are responsible for the observed methylation differences. However, it has been shown that MeHg exposure inhibits PON1 activity even after controlling for the major known genetic variants, suggesting a dominant epigenetic control mechanism for the enzyme. While we observed associations between DNA methylation and gene expression these associations may not be generalizable to our cohort. Another important limitation of our study is the use of adult reference DNA methylation data to impute white blood cell distribution, as adult blood might not accurately capture leukocyte distribution in childhood. However, we used a cord blood reference panel to estimate cell proportions at birth, including nucleated red blood cells, and the persistence of epigenetic modifications were robust to white blood cell composition adjustment using different panels for cord blood and child blood. We also lack mercury exposure information during early and mid-childhood or any pesticide exposure information and therefore the relative contribution of postnatal exposures cannot be determined. The study was observational in nature and confounding cannot be ruled out.54 and over 70% of the red-blood cell mercury is estimated to be methylmercury55. It is expected that this biomarker captured prenatal exposures occurring during critical windows of embryogenesis and fetal development. Although our sample size is moderate, this is the largest epigenome-wide study of prenatal mercury exposure conducted to date and the only one to prospectively test for persistence of epigenetic modifications at two time points during childhood. Our moderate sample size enabled us to evaluate sex-specific differences. Furthermore, this cohort has detailed anthropomorphic and demographic information that allowed us to control for many potential confounders and test for associations with cognitive test scores during early childhood. Detailed information on maternal diet also allowed us to control for fish intake, previously shown to be beneficial for children’s cognitive development, minimizing the possibility for negative confounding8. The analyses of regional and individual CpG sites methylation variability is also an important strength of the present study. Lastly, the potential for functional changes in DNA methylation with gene expression was evaluated in a separate set of cord blood samples further supporting our findings.The present study has many strengths that include the use of second trimester maternal red-blood cell mercury concentrations, an unbiased biomarker with an estimated half-life of 72 daysPON1 serves as a potential target of toxic environmental exposures particularly during fetal development and childhood. Furthermore, DNA methylation of PON1 could modulate the association between prenatal mercury exposure, cognitive development and other health outcomes in children. Epigenetic modifications leading to functional genomic changes could help explain heterogeneous findings observed for prenatal mercury exposure and cognitive development in different populations. This will also provide tools to design future targeted public health interventions.This study adds to the growing body of evidence that suggests that 56. This cohort was recruited between 1999 and 2002 during their first prenatal visit at Atrius Harvard Vanguard Medical Associates, a large multispecialty group practice. Mothers provided written informed consent at study recruitment and at every follow-up visit. All study protocols were reviewed and carried out in accordance with guidelines approved by the human subjects committee of Harvard Pilgrim Health Care. Eligibility criteria included fluency in English, gestational age less than 22 weeks at the first prenatal visit, and singleton pregnancy. Additional details of the cohort have been published elsewhere56. Of the total 2,128 mother-infants pairs in the cohort, we measured prenatal mercury exposure from 1019. Of these, we included 321 with available measurements of cord blood DNA methylation. Persistence of the epigenetic modifications were evaluated in 75 children with available whole blood DNA methylation measurements from early childhood (2.9 to 4.9 years) and 291 children with blood samples from mid-childhood (6.7 to 10.5 years) as well as prenatal mercury concentrations. Early and mid-childhood participants were not restricted to participants with cord blood samples and therefore are not mutually inclusive. Of the 321 infants eligible for analyses with cord blood samples, 70 were included for early childhood analyses and 160 in the mid-childhood analyses. The number of participants sampled in both early and mid-childhood was 43.Mothers and children were participants in Project Viva, a prospective pre-birth cohort study conducted in Massachusetts, USA8. Briefly, at the second trimester visit we obtained a maternal blood sample that was centrifuged to separate plasma from erythrocytes. Erythrocyte aliquots were stored at −70 °C and analyzed for total mercury by using the Direct Mercury Analyzer 80 . Results are reported as mercury content in the original red blood cell sample. The detection limit was 0.5 ng/g of sample, and the percentage recovery for standards ranged from 90 to 110%. At mid-pregnancy, participants also completed a semi-quantitative food frequency questionnaire that we previously calibrated against erythrocyte levels of elongated n-3 fatty acids57. Participants self-reported on the consumption of fish with four questions: “canned tuna fish (3–4 oz.)”; “shrimp, lobster, scallops, clams (1 serving)”; “dark meat fish, e.g. mackerel, salmon, sardines, bluefish, swordfish (3–5 oz.)”; and “other fish, e.g. cod, haddock, halibut (3–5 oz.)”, during the preceding 3 months. Six frequency response options ranged from “never/less than 1 per month” to “1 or more servings per day.” We combined responses to estimate average total fish intake in servings per week.Sample collection and exposure assessment for prenatal mercury exposure in this cohort has been previously described in detailTrained medical personnel obtained umbilical cord blood samples immediately upon delivery storing them in a dedicated refrigerator at 4 °C and transported to a central location within 24 hours of sample collection. Similarly, whole blood samples collected during early, and mid-childhood were stored at 4 °C and transported to the central storage location for sample processing. Subsequently, trained laboratory staff processed the samples on the same day of arrival, and DNA was extracted using the Qiagen Puregene Kit . Aliquots were then stored at −80 °C until analysis.Buffy coat DNA was sodium bisulfite converted using the EZ DNA Methylation-Gold Kit . Samples were allocated to plates using a two-stage algorithm by randomizing 12 samples to each chip and then randomly assigning eight chips to each of the 15 plates used to ensure balance by sex across chips and plates. Samples were shipped to Illumina Inc., and analyzed using the Infinium Human Methylation450 BeadChip  following standard manufacturer’s protocols. The Human Methylation450 BeadChip measures DNA methylation at >485,000 CpG sites simultaneously at a single nucleotide resolution, covering 99% of the RefSeq genes.minfi package in R58. Samples were excluded as potentially miss-labelled if they were mismatches on sex (n = 6), genotype (n = 6) or were deemed to be low in quality (n = 12). Technical replicates were also excluded from the analysis (n = 40). Correlation coefficients for individual probes among all technical replicates ranged from 0.98 to 1. We excluded individual probes if they had non-significant detection P-values (P > 0.05) for more than 1% of the samples. Additionally, non-CpG probes (i.e. rs and ch), probes in X and Y chromosomes, SNP-associated probes at either the single base extension or within the target region were removed for SNPs that have a minor-allele frequency of >5%. Previously identified non-specific and cross-reactive probes within the array along with polymorphic CpG loci were also excluded from the analysis59. After quality control on the probes, the total number of autosomal CpGs left for analysis was 384,349 loci for all the cord blood samples. Background correction and dye-bias equalization was performed via the normal-exponential out-of-band (noob) correction method60. Finally, a β-mixture quantile intra sample normalization procedure (BMIQ) was applied to the resulting data to reduce the potential bias that can arise from type2 probes59. For each CpG site, methylation is reported as average β-value = M/(M + U + ε), where M and U represent the average fluorescence intensity from the probe corresponding to the methylated and unmethylated target CpG and ε = 100 is a small quantity to protect against division by zero. Thus, the average β-value is an interval scaled quantity between zero and one interpreted as the fraction of DNA molecules whose target CpG is methylated.We processed raw methylation image files using the 61 to correct for Batch effects from plate and other potential sources of technical variability in methylation measurements. We visually inspected the effectiveness of adjustment for batch using the four main principal components before and after batch adjustment. Strip plots of control probes were visually examined for bisulfite conversion and specificity. Density plots for the β-values were examined across samples at each normalization step. Methylation values on the β-scale were logit transform to M-values as previously described to be more appropriate for differential analysis of DNA methylation62. All tables and results are presented on the β-value scale to ease interpretability.We used ComBatr > 0.90) with verbal and full-scale intelligence quotient of the Wechsler Intelligence Scale for Children-III63. The Wide Range Assessment of Visual Motor Abilities (WRAVMA) evaluates domains of visual motor development and is moderately correlated with intelligence quotient (r~0.60)64.At the early childhood visit, trained research personnel administered the Peabody Picture Vocabulary Test (PPVT) and the Wide Range Assessment of Visual Motor Abilities (WRAVMA) cognitive tests. The PPVT evaluates receptive vocabulary for children age 2 or older based on a national reference sample and it is strongly correlated (65 from isolated cell types. To estimate cell types composition in cord blood we used a reference panel of nucleated cells isolated from cord blood (leukocytes and nucleated red blood cells)66 and an adult leukocyte reference panel for blood samples collected in early or mid-childhood as implemented in minfi67. We conducted a principal component analysis in the resulting 384,349 CpGs across all cord blood samples and used univariate linear regression models to explore associations among each of the top 30-principal components with phenotypic and technical covariates using the EnMix Bioconductor package of R68. All analyses were carried out using the R statistical package, version 3.2.3 (www.r-project.org/).We calculated means and standard deviations (SD) or sample size and percentage for all covariates to describe the study population during the three time points: birth, early childhood (2.9 to 4.9 years) and mid-childhood (6.7 to 10.5 years). White blood cell composition was estimated from DNA methylation measurements using the Houseman projection method69. This regional analysis method was chosen over other available methods because it has an agnostic approach to both genomic annotation and direction of effect estimates. Briefly, this package first fits individual linear regression models using limma for each CpG and subsequently applies a Gaussian kernel smoothing function to test statistics grouping significant probes based on a maximum distance of λ = 1,000 base pairs. Significance testing among DMRs are adjusted for multiple comparisons using an FDR < 0.05. Regional analyses were performed in cord blood for all of the samples and also stratified by sex while adjusting for child gestational age at delivery, sex (if not stratified), and estimated nucleated cell types in cord blood . As well as maternal age, race/ethnicity, mean weekly fish intake during pregnancy, pre-pregnancy BMI, smoking during pregnancy, parity and college education.We examined the association of prenatal mercury exposure with differentially methylated regions (DMRs) in cord blood using the R Bioconductor package DMRcate2-transformed mercury exposure we used robust linear models to estimate the association of each CpG in the region as well as mean methylation levels of all CpG sites in the DMR relative to maternal RBC-Hg concentrations adjusting for potential confounders. Persistence of the epigenetic changes was evaluated in early and mid-childhood blood DNA using similar robust linear models. Multivariate linear models were adjusted for the same covariates as the cord blood models with the exceptions that child race was used instead of maternal race, white blood cell composition was estimated from whole blood DNA methylation using an adult reference panel, and models were further adjusted by age of the child in days at the time of the blood draw. Associations were considered to be persistent with P < 0.05.Among significant DMRs found relative to logM-value scale as the dependent variable and log2-transformed prenatal mercury exposure as the main predictor while adjusting for the same covariates as in the regional analyses. CpG-by-CpG analyses were further stratified by infant sex to evaluate sex-specific epigenetic disruption at individual sites. The genomic inflation factor (λ) for the overall analysis was 0.99 indicating of no major systemic biases. Statistical significance for the CpG-by-CpG analyses was evaluated using a Bonferroni adjusted level of significance (P < 1.3 × 10−7). Although the epigenome-wide analysis was performed on the M-value scale, all estimates are reported as percent change in methylation along with the corresponding P-values from robust linear regression models performed on the β-value scale, for ease of interpretability.We evaluated methylation differences at individual CpG sites in cord blood relative to maternal RBC-Hg concentrations using robust linear regression with heteroskedasticity-consistent estimators to model the methylation levels of each individual CpG on the P < 1.3 × 10−7 in cord blood analyses to subsequent analyses for blood samples collected during early and mid-childhood to evaluate the persistence of associations using multivariate robust linear regression models adjusting for covariates. We considered P < 0.05 as indicating the persistence of epigenetic alterations in early or mid-childhood for the CpG-by-CpG analyses.We carried forward individual loci with a 22.To evaluate if the epigenetic alterations observed relative to prenatal mercury exposure are related to functional genomic changes in cord blood (i.e. expression), we used two publicly available datasets from an independent birth cohort found in the Gene Expression Omnibus (GEO) repository. Specifically, we identified DNA methylation data from 38 cord blood samples that used the Illumina Human Methylation450 array (GSE62924) and paired them with gene expression profiles from the same subjects (GSE48355) measured using the Affymetrix Human Gene 2.0 ST array. The study design, methods, sample collection and processing for this birth cohort have been previously describedWe estimated correlation coefficients between gene expression and DNA methylation at CpGs within the DMR and at individual sites observed to be differentially methylated in cord blood relative to prenatal mercury exposure in our data. For the DMR found to be associated with prenatal mercury exposure, correlation coefficients between expression and mean methylation levels of the region as well as at individual CpGs within the region were tested. Correlation coefficients along with 95% Confidence Intervals (95% CIs) were used to estimate the association.Linear regression models were used to estimate the association between differentially methylated DMRs or CpGs found in cord blood and cognitive test scores (PPVT and WRAVMA). Models were adjusted for maternal education at study enrollment, maternal PPVT scores, self-reported alcohol use during pregnancy, fetal growth , mean weekly fish intake during pregnancy, child age in days at the time of testing, sex (if not stratified), child race, parity and maternal smoking during pregnancy. Estimates are reported for every 10% increase in methylation levels along with 95% CIs.Supplemental Material


Here we present a candidate New World monkey model of ZIKV infection in pregnant marmosets that faithfully recapitulates human disease. ZIKV inoculation at the human-equivalent of early gestation caused an asymptomatic seroconversion, induction of type I/II interferon-associated genes and proinflammatory cytokines, and persistent viremia and viruria. Spontaneous pregnancy loss was observed 16–18 days post-infection, with extensive active placental viral replication and fetal neurocellular disorganization similar to that seen in humans. These findings underscore the key role of the placenta as a conduit for fetal infection, and demonstrate the utility of marmosets as a highly relevant model for studying congenital ZIKV disease and pregnancy loss.During its most recent outbreak across the Americas, Zika virus (ZIKV) was surprisingly shown to cause fetal loss and congenital malformations in acutely and chronically infected pregnant women. However, understanding the underlying pathogenesis of ZIKV congenital disease has been hampered by a lack of relevant  Aedes genus) arbovirus of the Flaviviridae family. Originally discovered in Uganda in 19471, previous outbreaks of ZIKV were largely sporadic across Southeast Asia and the equatorial African belts, but later spread east resulting in an outbreak in Yap Island in 2007, followed by epidemics in French Polynesia, New Caledonia, the Cook Islands, and Easter Island in 2013 and 20142. The virus emerged in the Caribbean and South America in late 2014, persisting currently across the Western Hemisphere as of 2017 (https://www.cdc.gov/zika/geo/index.html). The virus is geographically spread as a result of human travel from endemic regions, alongside human-to-human transmission via sexual intercourse, with blood transfusions, and via vertical maternal-fetal transmission5.Zika virus (ZIKV) is a mosquito-borne 11. Rhesus, pigtail, and cynomolgus macaque and squirrel monkey models of ZIKV infection have also been developed16. Acute ZIKV infection in these nonhuman primate (NHP) models early in the course of the pregnancy have been shown to result in an average spontaneous abortion rate of 38% , with one study in a single pigtailed macaque demonstrating impaired neurodevelopment associated with ZIKV infection of the fetal brain and neuroprogenitor cells9. However, there are several limitations to existing NHP models of viral congenital infections, including long lengths of gestation, limited mating periods associated with long intervals of lactation-induced or seasonal ovulatory suppression between pregnancies, and a large body size that makes higher biocontainment housing and testing of limited-quantity vaccines or therapeutics challenging.A number of animal models of ZIKV infection have been described to date. These include several murine models in which ZIKV-associated complications in pregnant females required the use of immunodeficient animals with defects in the interferon-related signaling pathwaysCallithrix jacchus), a rat-sized, neotropical primate, as a relevant and convenient model for investigating viral pathogenesis17. The common marmoset model has been shown to reproduce the human illnesses caused by Lassa virus18, Ebola/Marburg virus19, and flavivirus (dengue virus and West Nile virus)21 infection. We also recently reported that the course of infection in ZIKV-infected male marmosets resembles that seen in acutely infected humans in many aspects22. Specifically, viral illness was subclinical, the virus persisted in body fluids such as semen and saliva for longer periods of time than in serum, and there were neutralizing antibody as well as characteristic interferon-associated host responses. Encouraged by these initial studies demonstrating that viral illness in male marmosets is akin to the human condition, we sought to determine whether infection of marmoset dams would more faithfully recapitulate maternal infection and CZS in humans when compared to murine models and provide key opportunities not afforded by other NHP models. As a nonhuman primate, the marmoset has a hemomonochorial placental architecture that develops early in pregnancy23, a situation much more similar to humans than that of the chorioallantoic placental architecture of the mouse that is only mature relatively late in gestation24. Moreover, we hypothesized that crucial mechanistic insights pertaining to placental infection and vertical transmission could be reached as a result of the unusual but well described characteristics of trophoblast and embryonic development in the common marmoset. Marmosets have a delayed period of embryogenesis relative to humans23, resulting in the marmoset undergoing critical stages of neural development in association with a relatively far more mature placenta . Here we report the outcomes of experimental infection with ZIKV in two pregnant marmoset dams, and the resultant impact on the placenta and developing fetus.In contrast, mounting evidence over the past decade has pointed to the common marmoset (5 plaque-forming units (PFU), followed by a second inoculation with the same dose 4 days later  with Brazil ZIKV strain SPH2015 (an Asian lineage strain first isolated in 2015) at estimated gestational days 79 (Dam 1) and 68 (dam 2) with a dose at 2.5 × 10ter Fig. . Fetusesa priori-determined specific intervals, including a pre-infection sample taken 5 days before infection, and for up to 30 days following inoculation  and persisted for 2 and 3 weeks respectively  in maternal lymph node, ocular, and renal tissues, as well as in placental tissues and fetal brain/cranium and abdominal tissues Fig. .Two days prior to fetal demise and expulsion, Dam 1 was confirmed by fetal sonogram to carry a live dichorionic, diamniotic twin gestation with normal amniotic fluid volumes and cardiac activity. The spontaneous demise and delivery of the twin fetuses and their placentae were discovered approximately 20–36 hours after expulsion (3 days post sonogram). Both of the aborted fetuses were friable and each had a measured biparietal diameter (BPD) that were 50% of the expected values based on the ultrasounds performed 2 and 4 days earlier; however, this is most likely due to cranial collapse and dolicocephaly typical in fetal tissue after prolonged (>4–6 hours) demise. One twin fetus was immediately placed in formalin for the histological analyses, while the other fetus was dissected for RNA viral load testing Fig. . The pla22. Thus, although marmosets are obligate carriers of multi-fetal gestations due to a polygenic set of synonymous and non-synonymous substitutions26, in a number of instances they have been shown to carry singleton fetuses successfully to term27. After confirming a continuing developing viable singleton gestation, dam 2 was infected with 2 ZIKV inoculums on days 68 and 72. A viable singleton gestation was confirmed on day 82 and subsequently demised with fetal expulsion on day 86. The single expulsed fetus and its placenta were immediately recovered; the head was formalin-fixed for histology while the body was dissected for RNA viral load testing , targeting positive-strand ZIKV RNA and its corresponding negative-strand replicative intermediate, established the presence of viral genomic RNA and active replication within placental tissue, respectively  compared to the replication (−) strand  and (−) strand probes, reserving the corresponding abdomen and thorax from the dam 2 fetus for viral load testing Figs  and 5. H31, close examination of fetal cranium and its contents from both dam 1 and dam 2 fetuses was made to characterize and anatomically localize cephalic viral infection and replication. The ophthalmic and cerebral regions of a frontally sectioned head of dam 2 fetus were examined . The original ZIKV inoculum as well as placenta and fetus from dam 1 retained the original phenylalanine residue at that position.ZIKV genome sequencing from tissues and body fluids, done in parallel from the original inoculum, was performed to identify mutations arising after infection. In the infected placenta and singleton aborted fetus from dam 2, a single consensus coding mutation (99.5% frequency) was identified in the matrix protein resulting in an amino acid change from phenylalanine to leucine (F252L) (Supplementary Table Whole blood from both the asymptomatic ZIKV-infected dams collected at post-infection days 2 (for only 1 of 2 dams), 7, and 30 were collected for transcriptome profiling by RNA-seq, and compared to whole blood samples drawn from the same animals prior to ZIKV inoculation. Each sample was sequenced in duplicate to increase statistical power. The average sequencing depth was 23.9 M reads per sample (±6.7 M reads)  at 2, 7, and 30 days post-infection relative to the uninfected baseline. Distinct from our previously published in male marmosets, in which maximal differential expression was observed at day 7 post-ZIKV infection35. As a result, murine models of ZIKV infection require IFN manipulation and are thus inherently limited in their potential for both translational relevance and mechanisms of congenital pathogenesis36. Here we show that ZIKV inoculation of pregnant marmosets causes a maternal asymptomatic infection and seroconversion similar to that seen in humans. This maternal infection is measured as prolonged viremia and viruria, and is accompanied by active viral replication in the placenta, with resultant fetal infection, a predominant neuropathology of the developing fetal brain and eye, and early demise. Although atypical for non-pregnant human cases, persistence of virus in serum and urine (i.e. viremia and viruria) beyond 14 days and as long as 8 months has been reported in ZIKV-infected pregnant women37, and is felt to be due to permissive replication in the placenta and/or fetus functioning as a viral reservoir29. In both inoculated dams, spontaneous abortion was observed 16–18 days post infection, with extensive productive viral infection in the placenta and fetal tissues documented by qRT-PCR, immunohistochemistry, and ISH for (+) and (−) ssRNA strands. We also visualized direct infection of neural progenitor cells associated with disorganized migration. The most straightforward explanation for the measured 50% expected biparietal diameter of the fetuses from dam 1 is cranial collapse following intrauterine demise; however, we also cannot rule out this finding as an early predictor of microcephaly since it is temporally and spatially accompanied by aberrant migration patterns which are hallmarks of secondary microcephaly. In 1 of the 2 inoculations, a spontaneous de novo mutation, F252L, shared in common with a ZIKV genome from a human case of microcephaly, was identified. Finally, maternal interferon-associated host responses and increased activity of proinflammatory cytokines were demonstrated at early as day 2 post-inoculation. Collectively, these findings reveal several intriguing and novel mechanistic insights pertaining to the potential roles of placental replication, mutation-driven viral adaptation, and maternal responses in ZIKV-mediated fetal pathogenesis.Rodents, including laboratory mice, are not a natural host of ZIKV infection because their IFN-driven immune responses can inhibit ZIKV replication38. The investigators documented a pregnancy loss rate during days 80–142 of 4.4%; our finding of 2 of 2 ZIKV-infected pregnancies resulting in spontaneous abortion is thus statistically significant (p = 0.011 by Fisher’s Exact Test). In comparison, the overall loss rate in experimentally ZIKV-infected macaques at a comparable stage of embryonic development is 38%, as compared to expected non-infected loss rates of approximately 4–10% . In humans, documented rates of pregnancy loss due to ZIKV across all 3 stages at time of infection are estimated to be ~3%, with 8% of completed pregnancies following infection during the first trimester resulting in ZIKV-associated birth defects4. However, due to inherent limitations in the studies of miscarriages, early and mid-gestation ZIKV-induced pregnancy loss has been likely underreported and largely limited to case reports and limited retrospective series39. We speculate that our observation of uniform pregnancy loss in our marmoset model is a highly significant novel finding of potential translational significance and worthy of prioritized further human studies and re-analysis of existing epidemiologic data40.The first trimester of pregnancy in both humans and marmosets is critical for appropriate development of the major organ systems, especially the brain and its associated neuroprogenitor cells. Gestational ages of 79–83 days (dam 1) and 68–72 days (dam 2) at the time of inoculation correspond to approximately 6.6–8.6 weeks in humans  increases in the concentrations of several proinflammatory molecules, including IFN-γ, which drives the type II interferon pathway. Interestingly, an antiviral response characterized by increased interferon and proinflammatory cytokine activity has been previously reported in association with ZIKV infection of primary human placental macrophages (Hofbauer) cells and cytotrophoblasts41. Here we observed extensive ZIKV infection of the placenta and fetus in the absence of overt inflammation and despite induction of a robust host antiviral response. This is consistent with ours and others recently described human placental pathogenesis47.Only rodents rendered deficient of type I interferon through experimental manipulations are highly susceptible to maternal-fetal transmission and resultant congenital disease48, and has been shown to be critical for maternal-fetal Ca2+ transport and fetal Ca2+ homeostasis in an aromatase knockout pregnant mouse model49. In this mouse model, decreased expression of TRPV6 was associated with estrogen deficiency, which increases risk of miscarriage50. Like TRPV6, LYP6 is highly expressed in placenta51, and was critical for normal neuroectodermal development by way of Wnt/β-catenin signaling in zebrafish embryos52. Further studies are needed to determine whether viral suppression of these highly expressed maternal placental genes plays a role in the spontaneous abortions and disrupted fetal neurodevelopment seen here in association with first-trimester ZIKV infection.Among the 31 candidate DEGs at day 7 post-infection, the decreased expression of TRPV6 and LYPD6 relative to baseline is especially intriguing. TRPV6 is highly expressed in placentaAn important point of consideration for applying our current findings to human congenital disease is an appreciation of the timeline of embryonic and placental development in marmoset monkeys versus humans. Marmosets have a delayed period of embryogenesis relative to humans Fig. , resulti15. Pigtail macaques exhibited neural injury with severe hypoplasia and asymmetry within the occipital-parietal lobes9, but did not manifest the diffuse effects on neuroprogenitor cells observed in both the marmoset and human cases. In contrast, neuronal disorganization in ZIKV-infected pregnant marmosets was observed during early gestation at the posterior and crown in what will later comprise the occipital–parietal lobes 39 without evidence of inflammation47. As is likely the case in humans, the placenta may be viewed as an effective “shuttle” to the fetus, functioning first as a reservoir and later as a source of fetal infection of susceptible cell types and tissues, including neuroprogenitor cells43. Nevertheless, in fetuses from both dam 1 and dam 2, the virus appears to have caused injury to the neocortex documented by histopathological examination and neuronal disorganization in the absence of localized inflammation, as also seen in mouse models11 and human cases44.Our findings herein summarily show that the marmoset model faithfully recapitulates key characteristics of CZS in humans that are not observed in other rodent nor NHP models. Notably, ZIKV-infected rhesus macaques exhibited no fetal CNS abnormalities with maternal infectionbes Fig. . We spec47. However, there is clear evidence of injury to the fetal neocortex as visualized by histopathological examination showing neuronal organization, mimicking findings in mice models of ZIKV vertical transmission as well as human cases44. This notion of the significance of placental mechanics and placental cell infection in the pathogenesis in CZS has been previously brought forward by a number of investigators53. It has long been supposed that shuttling of bloodborne ZIKV to the developing human fetus requires establishment of maternal blood in the intervillous space, which occurs approximately at 6–8 weeks post-conception (mean of roughly 42 days)  protein . Of note, the African strains used in the work of Bayer et al.45, where they failed to see robust ZIKV replication in primary human trophoblasts, did not demonstrate the S139N mutation. The virus from dam 2 had a further de novo F252L coding mutation shared in only 1 of 6 available microcephalic ZIKV genomes in humans33. The serendipitous finding of a F252L coding mutation is unlikely to be coincidental, as the mutation was not seen in any of the other 603 ZIKV genomes sequenced to date not associated with microcephaly (p = 0.0135). Further investigation of the potential functional significance of the F252L mutation is warranted.Contemporary ZIKV strains may have evolved to acquire the capacity for placental replication and fetal neuropathogenesis36. Given the capacity for the marmoset to complete multifetal gestations in 143 days and undergo repeat pregnancy within 10 days, this is an efficient model whose reproductive capacity over short time intervals rivals most rodent models. Second, these studies underscore crucial mechanistic insights pertaining to the role of the placenta in modulating (or failing to modulate) congenital infection and maternal-to-fetal transmission. Understanding the role of placental permissiveness in this process is of utmost crucial importance and reinforces the emerging notion of the placenta not as a de facto barrier, but as an active conduit for maternal and fetal communication during gestation. Thus, the identification of drugs not only to treat or prevent ZIKV infection of the fetus but also to prevent placental infection and viral transport may be critical to prevent the irreversible neuronal damage associated with CZS. Third and of potential crucial clinical importance, our findings demonstrate a high rate of pregnancy loss with maternal ZIKV infection and placental and fetal transmission. These observations should prompt reexamination of human epidemiologic data and provide additional endpoints in prospective studies as ZIKV continues to emerge in newly endemic communities in Texas, Florida, and other at-risk regions of the Americas.We are enthused by both the potential relevance and significance of our findings employing pregnant marmosets. First, they show that the marmoset faithfully recapitulates all crucial aspects of human CZS and does not require the exogenous manipulation of interferons, necessary in murine modelsAnimal studies were conducted at the Southwest National Primate Research Center (SNPRC), an AAALAC-accredited program. All studies were reviewed and approved by the local Institutional Animal Care and Use Committee (IACUC) and Biohazard Committee.5 plaque forming units (PFU) of the Brazil ZIKV strain SPH2015 diluted in saline (GenBank accession number KU321639)55, followed by a second identical inoculation 4 days later. The gestational age of the embryos at the time of inoculation was determined by crown-rump length, which we have previously demonstrated to provide a reliable gestational age to within +/−3 days56. Embryonic growth was tracked by ultrasonography performed on unsedated animals. For dam 1, ultrasound exams took place on days 80, 88, and 93, confirming two live embryos with biparietal diameters as expected for those ages. Ultrasounds on dam 2 were on days 78 and 82, confirming one live embryo. In each case, the number of embryos was consistent from pre-dosing screening through the infection period.Two pregnant dams  were inoculated intramuscularly with 0.25 mL containing with 2.5 × 1057. Urine, blood, and saliva were sampled and serially monitored for ZIKV RNA by qPCR testing per a pre-established schedule , within or exceeding the published range of weight gain for pregnant dams at mid-term (0.39–0.71 grams/day)ule Fig. . For theule Fig. .5. By standard curve analysis, the estimated limit of detection for the qRT-PCR assay was ~15 RNA copies per mL.The course of infection in inoculated marmosets was monitored by determination of ZIKV RNA loads (expressed as RNA copies/mL) in infected body fluids  and placental and fetal tissues. ZIKV RNA loads were calculated by generation of a standard curve, followed by quantitative RT-PCR testing for 45 cycles using published PCR conditions and primers targeting the envelope gene (ZIKV-1086/ZIKV-1162)2 incubator for 3 days, followed by addition of 3 mL of 1% agar and 0.004% neutral red in Eagle’s MEM and another 1–2 days of incubation until plaques were formed. An 80% or more reduction of the number of plaques compared to positive control wells inoculated with virus-diluent mixtures was considered neutralization, with serum titers reported as the highest dilution exhibiting 80% or more reduction.Plaque-reduction neutralization assay (PRNT) on longitudinally collected marmoset sera was performed by the California Department of Public Health, using a protocol adapted from that used by the US Centers for Disease Control and Prevention (CDC) for confirmatory ZIKV testing in patients. Briefly, 100 plaque forming units (PFU) of ZIKV (Brazilian SPH2015 strain) were mixed with equal volumes of serial 2-fold dilutions of inactivated marmoset sera and incubated for 1 hr at 36 °C, followed by inoculation and adsorbing to a monolayer culture of Vero cells for 1 hr at 36 °C. After addition of 3 mL of 0.6% agar in Eagle’s Minimal Essential Medium (MEM), plates were placed in a 36 °C, 5% CO58. Data was scanned for ZIKV reads using an NCBI BLASTn database constructed from all known ZIKV sequences as of October 2017, using an E-value significance threshold of 1 × 10−5. Consensus ZIKV genomes were assembled using Geneious (v10.2.2) by mapping preprocessed ZIKV reads to reference genome KU321639.Nucleic acid was extracted from 50 μg of placenta or fetus lysate, 50 μL of body fluid sample , or 100 μL of viral culture inoculum using the automated Qiagen EZ1 instrument , followed by treatment with Turbo DNase . cDNA was obtained by reverse transcription using a 1:5 mix of random hexamer and short spiked primers targeting ZIKV genomes (ref). Next-generation sequencing (NGS) libraries were constructed using the Nextera XT kit , followed by single-end, 150 base pair (bp) sequencing on an Illumina MiSeq or HiSeq. 2500 instrument. Raw reads were preprocessed by adapter trimming and removal of low-quality, low-complexity sequences as previously described39. Briefly, 4 µM sections were deparaffinized and subjected to antigen retrieval as per manufacturer’s instructions . A previously designed probe set of complementary oligos against either strand, covalently bound to adapter sequences was then applied39. The ZIKV sequence specific probe sets consisted of 87 probe pairs requiring hybridization of adjacent matching genome sites for adapter probe hybridization and amplification. DAB signal was generated after a series of amplification probes as per manufacturer’s instructions. Placenta were also stained with 4G2 antibody against flavivirus protein E (Millipore) as previously described42. Vero cells  infected with contemporaneous ZIKV were used as a positive control. Cells were infected with first passage ZIKV HN16 (GenBank: KX928077.1) isolated from traveler returning to the USA from Honduras. Following 6 days of in vitro infection and replication, scraped cells were formalin fixed in a syringe, then paraffin embedded. Bright field slides were examined under low and medium power using a Nikon Eclipse 90i microscope . Minor adjustment for contrast and background levels were made using NIS Elements Viewer 4.20 (Nikon) and Adobe Photoshop CC.A full sagittal section was mounted of one dichorionic fetus from dam 1, and frontal and coronal sections of the singleton dam 2 pregnancy were formalin-fixed and paraffin-embedded. Sections were H&E stained to assess for localized inflammation, as well as to visualize and identify tissues within the fields. Serial sections of all marmoset tissues were further probed for ZIKV viral genome using RNAscope ZIKV probes  for both + (stable) and − (active replicating) RNA strands to determine subcellular localization of viral establishment and active replication. Probing utilized established methodologiesPlasma samples were analyzed for marmoset cytokines and chemokines by Luminex using established protocols for New World primates . Evaluation of analytes GRO-α (CSCL1), interferon alpha (IFN-α), IFN-γ, interleukin-1 beta (IL-1β), IL-22, monocyte chemoattractant protein 1 , macrophage migration inhibitory factor (MIF), monokine induced by gamma interferon , macrophage inflammatory protein 1-alpha , MIP-1β (CCL4), regulated on activation, normal T cell expressed and secreted , tumor necrosis factor-alpha (TNF-α), soluble CD40 ligand (sCD40L), soluble intercellular adhesion molecule 1 (sICAM-1), and vascular endothelial growth factor A (VEGF-A) were included in this assay. Only analytes quantifiable above the limit of detection are presented.Prior to inoculation, whole blood was drawn from the two age-/sex-matched healthy pregnant dams in the ZIKV study to use as internal controls in the transcriptome analysis. All marmosets studied here were from a single colony, thus increasing genetic similarities and decreasing environmental bias. 400 uL microliters of blood were drawn directly into RNAgard tubes (Biomatrica) for immediate RNA stabilization of intracellular RNA at time of collection. Total RNA was extracted using the Biomatrica Blood RNA Purification Kit (Biomatrica). The Ovation Human Blood RNA-Seq Kit  was used to generate RNA-seq libraries from 100 ng of input per sample (as measured using the Invitrogen Qubit RNA HS Assay Kit) according to the manufacturer’s protocol. Libraries were sequenced in duplicate as 100 bp paired-end runs on an Illumina HiSeq 2500 instrument.Callithrix jacchus Ensembl version 3.2.1), using STAR 2.559, and gene and transcript normalized counts were calculated by HTSeq version 0.6.060. Differential expression of genes was calculated using linear modeling using the Bioconductor EdgeR software package version 3.12.261, as implemented in the R programming language. Genes were considered to be differentially expressed when their fold change was >±2, p-value < 0.05, and adjusted p-value  < 0.1%. Pathway and network analyses of the transcriptome data were performed using Ingenuity Pathway Analysis (IPA) software (Qiagen). Marmoset transcriptome data has been submitted to the public National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) repository (accession numbers pending).Paired-end reads were mapped to the marmoset genome (The Texas Biomedical Research Institute IACUC approved all protocols for animal experiments.All p-values reported in this study are calculated using two-tailed Fisher’s Exact Test.Supplementary FilesSupplementary Table S3