Karen Vrijens

MicroRNAs as potential signatures of environmental exposure or effect: a systematic review.

Background: The exposome encompasses all life-course environmental exposures from the prenatal period onward that influence health. MicroRNAs (miRNAs) are interesting entities within this concept as markers and causation of disease. MicroRNAs are short oligonucleotide sequences that can interact with several mRNA targets. Objectives: We reviewed the current state of the field on the potential of using miRNAs as biomarkers for environmental exposure. We investigated miRNA signatures in response to all types of environmental exposure to which a human can be exposed, including cigarette smoke, air pollution, nanoparticles, and diverse chemicals; and we examined the health conditions for which the identified miRNAs have been reported (i.e., cardiovascular disease, cancer, and diabetes). Methods: We searched the PubMed and ScienceDirect databases to identify relevant studies. Results: For all exposures incorporated in this review, 27 miRNAs were differentially expressed in at least two independent studies. miRNAs that had expression alterations associated with smoking observed in multiple studies are miR-21, miR-34b, miR-125b, miR-146a, miR-223, and miR-340; and those miRNAs that were observed in multiple air pollution studies are miR-9, miR-10b, miR-21, miR-128, miR-143, miR-155, miR-222, miR-223, and miR-338. We found little overlap among in vitro, in vivo, and human studies between miRNAs and exposure. Here, we report on disease associations for those miRNAs identified in multiple studies on exposure. Conclusions: miRNA changes may be sensitive indicators of the effects of acute and chronic environmental exposure. Therefore, miRNAs are valuable novel biomarkers for exposure. Further studies should elucidate the role of the mediation effect of miRNA between exposures and effect through all stages of life to provide a more accurate assessment of the consequences of miRNA changes. Citation: Vrijens K, Bollati V, Nawrot TS. 2015. MicroRNAs as potential signatures of environmental exposure or effect: a systematic review. Environ Health Perspect 123:399–411; http://dx.doi.org/10.1289/ehp.1408459

In Utero Fine Particle Air Pollution and Placental Expression of Genes in the Brain-Derived Neurotrophic Factor Signaling Pathway: An ENVIRONAGE Birth Cohort Study.

Background Developmental processes in the placenta and the fetal brain are shaped by the same biological signals. Recent evidence suggests that adaptive responses of the placenta to the maternal environment may influence central nervous system development. Objectives We studied the association between in utero exposure to fine particle air pollution with a diameter ≤ 2.5 μm (PM2.5) and placental expression of genes implicated in neural development. Methods Expression of 10 target genes in the brain-derived neurotrophic factor (BDNF) signaling pathway were quantified in placental tissue of 90 mother–infant pairs from the ENVIRONAGE birth cohort using quantitative real-time polymerase chain reaction. Trimester-specific PM2.5 exposure levels were estimated for each mother’s home address using a spatiotemporal model. Mixed-effects models were used to evaluate the association between the target genes and PM2.5 exposure measured in different time windows of pregnancy. Results A 5-μg/m3 increase in residential PM2.5 exposure during the first trimester of pregnancy was associated with a 15.9% decrease [95% confidence interval (CI): –28.7, –3.2%, p = 0.015] in expression of placental BDNF at birth. The corresponding estimate for synapsin 1 (SYN1) was a 24.3% decrease (95% CI: –42.8, –5.8%, p = 0.011). Conclusions Placental expression of BDNF and SYN1, two genes implicated in normal neurodevelopmental trajectories, decreased with increasing in utero exposure to PM2.5. Future studies are needed to confirm our findings and evaluate the potential relevance of associations between PM2.5 and placental expression of BDNF and SYN1 on neurodevelopment. We provide the first molecular epidemiological evidence concerning associations between in utero fine particle air pollution exposure and the expression of genes that may influence neurodevelopmental processes. Citation Saenen ND, Plusquin M, Bijnens E, Janssen BG, Gyselaers W, Cox B, Fierens F, Molenberghs G, Penders J, Vrijens K, De Boever P, Nawrot TS. 2015. In utero fine particle air pollution and placental expression of genes in the brain-derived neurotrophic factor signaling pathway: an ENVIRONAGE Birth Cohort Study. Environ Health Perspect 123:834–840; http://dx.doi.org/10.1289/ehp.1408549

DFNA5: hearing impairment exon instead of hearing impairment gene?

Background: Three mutations in the DFNA5 gene have been described in three families with autosomal dominant non-syndromic hearing impairment. Although these mutations are different at the genomic DNA level, they all lead to skipping of exon 8 at the mRNA level. We hypothesise that hearing impairment associated with DFNA5 is caused by a highly unusual mechanism, in which skipping of one specific exon leads to disease that is not caused by other mutations in this gene. We hypothesise that this represents a very specific “gain of function” mutation, with the truncated protein exerting a deleterious new function. Methods: We performed transfection experiments in mammalian cell lines (HEK293T and COS-1) with green fluorescent protein (GFP) tagged wildtype and mutant DFNA5 and analysed cell death with flow cytometry and fluorescence microscopy. Results: Post-transfection death of HEK293T cells approximately doubled when cells were transfected with mutant DFNA5–GFP compared with wildtype DFNA5–GFP. Cell death was attributed to necrotic events and not to apoptotic events. Conclusion: The transfection experiments in mammalian cell lines support our hypothesis that the hearing impairment associated with DFNA5 is caused by a “gain of function” mutation and that mutant DFNA5 has a deleterious new function.

Mice lacking Dfna5 show a diverging number of cochlear fourth row outer hair cells

A complex mutation in DFNA5, resulting in exon 8 skipping, causes autosomal dominant hearing impairment, which starts in the high frequencies between 5 and 15 years of age and progressively affects all frequencies. To study its function in vivo, Dfna5 knockout mice were generated through the deletion of exon 8, simultaneously mimicking the human mutation. To test the hearing impairment, frequency-specific Auditory Brainstem Response (ABR) measurements were performed at different ages in two genetic backgrounds (C57Bl/6J and CBA/Ca), but no differences between Dfna5-/- and Dfna5+/+ mice could be demonstrated. Morphological studies demonstrated significant differences in the number of fourth row outer hair cells between Dfna5-/- mice and their wild-type littermates. These results were obtained in both genetic backgrounds, albeit with opposite effects. In contrast to the results obtained in Dfna5-/- zebrafish, we did not observe different UDP-glucose dehydrogenase and hyaluronic acid levels in Dfna5-/- mice when compared to Dfna5+/+ mice.

The DFNA5 gene, responsible for hearing loss and involved in cancer, encodes a novel apoptosis-inducing protein

DFNA5 was first identified as a gene causing autosomal dominant hearing loss (HL). Different mutations have been found, all exerting a highly specific gain-of-function effect, in which skipping of exon 8 causes the HL. Later reports revealed the involvement of the gene in different types of cancer. Epigenetic silencing of DFNA5 in a large percentage of gastric, colorectal and breast tumors and p53-dependent transcriptional activity have been reported, concluding that DFNA5 acts as a tumor suppressor gene in different frequent types of cancer. Despite these data, the molecular function of DFNA5 has not been investigated properly. Previous transfection studies with mutant DFNA5 in yeast and in mammalian cells showed a toxic effect of the mutant protein, which was not seen after transfection of the wild-type protein. Here, we demonstrate that DFNA5 is composed of two domains, separated by a hinge region. The first region induces apoptosis when transfected in HEK293T cells, the second region masks and probably regulates this apoptosis inducing capability. Moreover, the involvement of DFNA5 in apoptosis-related pathways in a physiological setting was demonstrated through gene expression microarray analysis using Dfna5 knockout mice. In view of its important role in carcinogenesis, this finding is expected to lead to new insights on the role of apoptosis in many types of cancer. In addition, it provides a new line of evidence supporting an important role for apoptosis in monogenic and complex forms of HL.

Air pollution-induced placental epigenetic alterations in early life: a candidate miRNA approach

ABSTRACT Particulate matter (PM) exposure during in utero life may entail adverse health outcomes in later-life. Air pollutions adverse effects are known to alter gene expression profiles, which can be regulated by microRNAs (miRNAs). We investigate the potential influence of air pollution exposure in prenatal life on placental miRNA expression. Within the framework of the ENVIRONAGE birth cohort, we measured the expression of six candidate miRNAs in placental tissue from 210 mother-newborn pairs by qRT-PCR. Trimester-specific PM2.5 exposure levels were estimated for each mothers home address using a spatiotemporal model. Multiple regression models were used to study miRNA expression and in utero exposure to PM2.5 over various time windows during pregnancy. The placental expression of miR-21 (−33.7%, 95% CI: −53.2 to −6.2, P = 0.022), miR-146a (−30.9%, 95% CI: −48.0 to −8.1, P = 0.012) and miR-222 (−25.4%, 95% CI: −43.0 to −2.4, P = 0.034) was inversely associated with PM2.5 exposure during the 2nd trimester of pregnancy, while placental expression of miR-20a and miR-21 was positively associated with 1st trimester exposure. Tumor suppressor phosphatase and tensin homolog (PTEN) was identified as a common target of the miRNAs significantly associated with PM exposure. Placental PTEN expression was strongly and positively associated (+59.6% per 5 µg/m³ increment, 95% CI: 26.9 to 100.7, P < 0.0001) with 3rd trimester PM2.5 exposure. Further research is required to establish the role these early miRNA and mRNA expression changes might play in PM-induced health effects. We provide molecular evidence showing that in utero PM2.5 exposure affects miRNAs expression as well as its downstream target PTEN.

Human hereditary hearing impairment: mouse models can help to solve the puzzle

Mouse models are important research tools in the discovery of genes for syndromic as well as non-syndromic forms of hearing impairment. The similarity between the human and mouse inner ear, as well as the high homology between the human and mouse genome, make the mouse an excellent tool for genetic research on hearing loss. Over the past ten years, multiple mouse models for human hereditary hearing loss have been identified. The current review discusses the models according to the biological process in which they are involved. We review mouse models for genes involved in acellular matrix composition, hair bundle formation and maintenance, outer hair cell electromotility, the hair cell ribbon synapse and auditory nerve transmission, regulation of cochlear homeostasis and regulation of transcription. We have opted to put most emphasis on mouse models for non-syndromic forms of human hearing loss. However, we also discuss advances made in the field of age related hearing impairment by the use of mouse models and modifiers of hearing loss genes identified in mice.

Lower Placental Leptin Promoter Methylation in Association with Fine Particulate Matter Air Pollution during Pregnancy and Placental Nitrosative Stress at Birth in the ENVIRONAGE Cohort.

Background: Particulate matter with a diameter ≤ 2.5 μm (PM2.5) affects human fetal development during pregnancy. Oxidative stress is a putative mechanism by which PM2.5 may exert its effects. Leptin (LEP) is an energy-regulating hormone involved in fetal growth and development. Objectives: We investigated in placental tissue whether DNA methylation of the LEP promoter is associated with PM2.5 and whether the oxidative/nitrosative stress biomarker 3-nitrotyrosine (3-NTp) is involved. Methods: LEP DNA methylation status of 361 placentas from the ENVIRONAGE birth cohort was assessed using bisulfite-PCR-pyrosequencing. Placental 3-NTp (n = 313) was determined with an ELISA assay. Daily PM2.5 exposure levels were estimated for each mother’s residence, accounting for residential mobility during pregnancy, using a spatiotemporal interpolation model. Results: After adjustment for a priori chosen covariates, placental LEP methylation was 1.4% lower (95% CI: –2.7, –0.19%) in association with an interquartile range increment (7.5 μg/m3) in second-trimester PM2.5 exposure and 0.43% lower (95% CI: –0.85, –0.02%) in association with a doubling of placental 3-NTp content. Conclusions: LEP methylation status in the placenta was negatively associated with PM2.5 exposure during the second trimester, and with placental 3-NTp, a marker of oxidative/nitrosative stress. Additional research is needed to confirm our findings and to assess whether oxidative/nitrosative stress might contribute to associations between PM2.5 and placental epigenetic events. Potential consequences for health during the neonatal period and later in life warrant further exploration. Citation: Saenen ND, Vrijens K, Janssen BG, Roels HA, Neven KY, Vanden Berghe W, Gyselaers W, Vanpoucke C, Lefebvre W, De Boever P, Nawrot TS. 2017. Lower placental leptin promoter methylation in association with fine particulate matter air pollution during pregnancy and placental nitrosative stress at birth in the ENVIRONAGE cohort. Environ Health Perspect 125:262–268; http://dx.doi.org/10.1289/EHP38

Placental Nitrosative Stress and Exposure to Ambient Air Pollution During Gestation: A Population Study

The placenta plays a crucial role in fetal growth and development through adaptive responses to perturbations of the maternal environment. We investigated the association between placental 3-nitrotyrosine (3-NTp), a biomarker of oxidative stress, and exposure to air pollutants during various time windows of pregnancy. We measured the placental 3-NTp levels of 330 mother-newborn pairs enrolled in the Environmental Influence on Ageing in Early Life (ENVIRONAGE) Study, a Belgian birth cohort study (2010-2013). Daily concentrations of particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5), black carbon (BC), and nitrogen dioxide were interpolated for each mothers residence using a spatiotemporal interpolation method. Placental 3-NTp levels, adjusted for covariates, increased by 35.0% (95% confidence interval (CI): 13.9, 60.0) for each interquartile-range increment in entire-pregnancy PM2.5 exposure. The corresponding estimate for BC exposure was 13.9% (95% CI: -0.21, 29.9). These results were driven by the first (PM2.5: 29.0% (95% CI: 4.9, 58.6); BC: 23.6% (95% CI: 4.4, 46.4)) and second (PM2.5: 39.3% (95% CI: 12.3, 72.7)) gestational exposure windows. This link between placental nitrosative stress and exposure to fine particle air pollution during gestation is in line with experimental evidence on cigarette smoke and diesel exhaust exposure. Further research is needed to elucidate potential health consequences experienced later in life through particle-mediated nitrosative stress incurred during fetal life.

Ozzy ,a Jag1 vestibular mouse mutant, displays characteristics of Alagille syndrome

The mouse mutant Ozzy, originating from an ENU-mutagenesis programme, displays a head bobbing phenotype. We report here that Ozzy mice show a clear deficit in vestibulo-ocular reflex (VOR). Micro-CT scanning of the inner ears showed narrowing and truncations of at least one of the semicircular canals and loss of the ampullae. Frequency-specific auditory-evoked brainstem response (ABR) tests revealed a slight threshold increase in the middle frequency range compared to wild-type littermates. Linkage analysis localised the gene in a 5.5-cM region on chromosome 2. Subsequently, a 499 T-->A missense mutation was identified in Jag1, leading to a substitution of an evolutionary conserved tryptophane (W167R). Mutations in the human homologue of Jag1 cause Alagille syndrome (AGS), an autosomal dominant disorder associated with liver, heart, eye and skeletal abnormalities, accompanied by a characteristic facies. In human patients, it occasionally affects other organ systems like the kidney or the inner ear. Liver disease is the main diagnostic factor for AGS. Ozzy mice showed significantly less intrahepatic bile ducts than wild-type littermates. Thirty-seven percent of Ozzy mice showed heart defects. No eye or vertebral abnormalities could be detected. In conclusion, Ozzy mice show two of the major and one minor characteristic of AGS.