Functional DNA methylation signatures for autism spectrum disorder genomic risk loci: 16p11.2 deletions and CHD8 variants.

BACKGROUND: Autism spectrum disorder (ASD) is a common and etiologically heterogeneous neurodevelopmental disorder. Although many genetic causes have been identified (> 200 ASD-risk genes), no single gene variant accounts for > 1% of all ASD cases. A role for epigenetic mechanisms in ASD etiology is supported by the fact that many ASD-risk genes function as epigenetic regulators and evidence that epigenetic dysregulation can interrupt normal brain development. Gene-specific DNAm profiles have been shown to assist in the interpretation of variants of unknown significance. Therefore, we investigated the epigenome in patients with ASD or two of the most common genomic variants conferring increased risk for ASD. Genome-wide DNA methylation (DNAm) was assessed using the Illumina Infinium HumanMethylation450 and MethylationEPIC arrays in blood from individuals with ASD of heterogeneous, undefined etiology (n = 52), and individuals with 16p11.2 deletions (16p11.2del, n = 9) or pathogenic variants in the chromatin modifier CHD8 (CHD8+/-, n = 7). RESULTS: DNAm patterns did not clearly distinguish heterogeneous ASD cases from controls. However, the homogeneous genetically-defined 16p11.2del and CHD8+/- subgroups each exhibited unique DNAm signatures that distinguished 16p11.2del or CHD8+/- individuals from each other and from heterogeneous ASD and control groups with high sensitivity and specificity. These signatures also classified additional 16p11.2del (n = 9) and CHD8 (n = 13) variants as pathogenic or benign. Our findings that DNAm alterations in each signature target unique genes in relevant biological pathways including neural development support their functional relevance. Furthermore, genes identified in our CHD8+/- DNAm signature in blood overlapped differentially expressed genes in CHD8+/- human-induced pluripotent cell-derived neurons and cerebral organoids from independent studies. CONCLUSIONS: DNAm signatures can provide clinical utility complementary to next-generation sequencing in the interpretation of variants of unknown significance. Our study constitutes a novel approach for ASD risk-associated molecular classification that elucidates the vital cross-talk between genetics and epigenetics in the etiology of ASD.

NSD1 mutations generate a genome-wide DNA methylation signature.

Sotos syndrome (SS) represents an important human model system for the study of epigenetic regulation; it is an overgrowth/intellectual disability syndrome caused by mutations in a histone methyltransferase, NSD1. As layered epigenetic modifications are often interdependent, we propose that pathogenic NSD1 mutations have a genome-wide impact on the most stable epigenetic mark, DNA methylation (DNAm). By interrogating DNAm in SS patients, we identify a genome-wide, highly significant NSD1(+/-)-specific signature that differentiates pathogenic NSD1 mutations from controls, benign NSD1 variants and the clinically overlapping Weaver syndrome. Validation studies of independent cohorts of SS and controls assigned 100% of these samples correctly. This highly specific and sensitive NSD1(+/-) signature encompasses genes that function in cellular morphogenesis and neuronal differentiation, reflecting cardinal features of the SS phenotype. The identification of SS-specific genome-wide DNAm alterations will facilitate both the elucidation of the molecular pathophysiology of SS and the development of improved diagnostic testing.

EBV transformation and cell culturing destabilizes DNA methylation in human lymphoblastoid cell lines.

Recent research suggests that epigenetic alterations involving DNA methylation can be causative for neurodevelopmental, growth and metabolic disorders. Although lymphoblastoid cell lines have been an invaluable resource for the study of both genetic and epigenetic disorders, the impact of EBV transformation, cell culturing and freezing on epigenetic patterns is unknown. We compared genome-wide DNA methylation patterns of four white blood cell samples, four low-passage lymphoblastoid cell lines pre and post freezing and four high-passage lymphobastoid cell lines, using two microarray platforms: Illumina HumanMethylation27 platform containing 27,578 CpG sites and Agilent Human CpG island Array containing 27,800 CpG islands. Comparison of genome-wide methylation profiles between white blood cells and lymphoblastoid cell lines demonstrated methylation alterations in lymphoblastoid cell lines occurring at random genomic locations. These changes were more profound in high-passage cells. Freezing at low-passages did not have a significant effect on DNA methylation. Methylation changes were observed in several imprinted differentially methylated regions, including DIRAS3, NNAT, H19, MEG3, NDN and MKRN3, but not in known imprinting centers. Our results suggest that lymphoblastoid cell lines should be used with caution for the identification of disease-associated DNA methylation changes or for discovery of new imprinted genes, as the methylation patterns seen in these cell lines may not always be representative of DNA methylation present in the original B-lymphocytes of the patient.

An inversion inv(4)(p12-p15.3) in autistic siblings implicates the 4p GABA receptor gene cluster.

INTRODUCTION: We describe the case of two brothers diagnosed with autism who both carry a paracentic inversion of the short arm of chromosome 4 (46,XY, inv(4)(p12-p15.3)). We have determined that this inversion is inherited from an apparently unaffected mother and unaffected maternal grandfather. Methods/ RESULTS: Using fluorescence in situ hybridisation analysis and Southern blot hybridisation we identified the breakpoints. The proximal breakpoint (4p12) maps to a region containing a cluster of gamma-aminobutyric acid A (GABA(A)) receptor genes, and directly interrupts the GABRG1 gene, the distal-most gene of the cluster. We also identified an insertion/deletion polymorphism for a approximately 2 kb LINE1 (L1) element that occurs within intron 7 of GABRG1. Our genotype analysis amongst autism families indicated that the L1 deletion allele did not show increased transmission to affected individuals. No linkage disequilibrium was evident between the L1 and single nucleotide polymorphisms in adjacent GABA(A) receptor genes on 4p, where a recent study has identified significant association with autism. DISCUSSION: Despite this, the identification of an inversion breakpoint disrupting GABRG1 provides solid support for the genetic involvement of the short arm of chromosome 4 in the genetic aetiology of autism, and for the hypothesis of disrupted GABA neurotransmission in autism.

Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells.

The present study was designed to verify if human (h) Angiotensin II (Ang II) type-1 receptor (hAT1R) undergoes internalization, nuclear translocation, and de novo synthesis in primary culture of human aortic vascular smooth muscle cells (hVSMCs) and if overexpression of this receptor modulates sustained free cytosolic ([Ca]c) and nuclear ([Ca]n) calcium. 3-dimensional (3-D) confocal microscopy was used to monitor free intracellular Ca2+ and hAT1R-green fluorescence protein (GFP) fusion protein in cultured hVSMCs. Immunofluorescence studies showed the presence of hAT1R and the absence of hAT2R in normal hVSMCs. Using 3-D imaging technique, hAT1 receptors were localized at the sarcolemma and in the cytosolic and nuclear compartments. In native as well as in normal hAT1R or hAT1R-GFP overexpressing hVSMCs, Ang II (10(-9) and 10(-4) M) induced internalization and nuclear translocation of this type of receptor. The internalization of hAT1Rs is mediated via clathrin-coated pits and vesicles pathway. This phenomenon of trancellular trafficking of receptors was associated with an increase of hAT1R. The Ang II induced increase of hAT1R density was prevented by the protein synthesis inhibitor cycloheximide. Overexpression of hAT1R and hAT1R-GFP decreased both basal cytosolic and nuclear Ca2+. In normal hVSMCs and low hAT1R-GFP overexpressing hVSMCs, Ang II (10(-15) to 10(-4) M) induced a dose-dependent sustained increase of [Ca]c and [Ca]n with an EC50 near 5 x 10(-11) and 5 x 10(-9) M, respectively. Our results suggest that hAT1Rs are the predominant type of Ang II receptors in aortic hVSMCs and are present in the sarcolemma, the cytosolic and the nuclear compartments. Ang II rapidly induces hAT1R internalization, nuclear translocation, as well as nuclear de novo synthesis of this receptor. The hAT1R overexpression in hVSMCs modulates sustained [Ca]c and [Ca]n.

Function of the endothelin(B) receptor in cardiovascular physiology and pathophysiology.

One of the two receptors by which the potent vasoactive effects of endothelin (ET)-1 are mediated is the ET(B) receptor (ET(BR)), which is found in several tissues, but, more importantly from a cardiovascular point of view, on the endothelial cell. The endothelial cell also has the unique capability of releasing ET-1, as well as other factors, such as the endothelial-derived relaxing factors and prostacyclin, which counteract the myotropic effects of the peptide. The secretory and contractile responses to ET-1 rely on G-protein-coupled ET(BR)s, as well as ET(A)-G-protein-coupled receptor-like proteins. The mitogenic properties of ET-1 via ET(A) receptors (ET(AR)s) coupled to mitogen-activated protein kinases and tyrosine kinases on the vascular smooth muscle may occur in conjunction with the anti-apoptotic characteristics of the endothelial ET(BR)s. Interestingly, most of the relevant antagonists and agonists for both ET(AR)s and ET(BR)s have been developed by the pharmaceutical industry. This highlights the therapeutical potential of compounds that act on ET receptors. In normal as well as in physiopathological conditions, the ET(BR) plays an important role in the control of vascular tone, and must be taken into account when using ET receptor antagonists for the treatment of cardiovascular diseases. For the management of congestive heart failure, renal failure and primary pulmonary hypertension, the most recent literature supports the use of selective ET(AR) antagonists rather than mixed antagonists of ET(AR)s and ET(BR)s. Nonetheless, validation of this view will have to await the first clinical trials comparing the actions of ET(A) to mixed ET(A)/ET(B) receptor antagonists.

Occupational allergy to bumblebees: allergens of Bombus terrestris.

BACKGROUND: With the increase in commercial vegetable production in greenhouses, occupational sensitization to bumblebee venom is becoming more common. Studies using sera from subjects thus sensitized allow evaluation of the allergenic specificity of bumblebee sensitization. OBJECTIVE: The purposes of this study were to determine the degree of species group specificity of bumblebee venom allergens in sera of allergic patients and to investigate the structural basis of this specificity. METHODS: Allergens were purified from bumblebee venom, studied serologically by direct binding and inhibition techniques, and characterized by enzyme analysis and amino acid sequencing. Three-dimensional models of the phospholipases were constructed and analyzed. RESULTS: Bombus terrestris venom contains phospholipase A(2), venom protease, hyaluronidase, and acid phosphatase allergens. The protease and phospholipase A(2) allergens contain IgE-reactive epitopes that are different from those seen in Bombus pennsylvanicus, a North American species. Bumblebee phospholipase A(2) is only 53% identical to honeybee phospholipase A(2). The results of 3-dimensional modeling are consistent with the immunologic observations. CONCLUSIONS: Patients with primary bumblebee sensitization should be diagnosed and treated with venom from the appropriate species group of bumblebees. Bumblebee venom phospholipase A(2) and protease are antigenically distinct from honeybee venom proteins. There are significant species group-specific epitopes on bumblebee venom proteins.

Endothelin-1 regulates cytosolic and nuclear Ca2+ in human endocardial endothelium.

Endocardial endothelium is far less studied than its systemic and pulmonary counterparts and its role in cardiac function is not well known. In the study reported here, we succeeded in isolating and culturing endocardial endothelial cells (EECs) from 21-week-old human fetal heart and verified whether endothelin-1 (ET-1) receptors are present on these cells and whether their activation regulates cytosolic ([Ca]c) and nuclear calcium ([Ca]n). Using fluo-3 Ca2+ measurement and three-dimension confocal microscopy techniques, superfusion of fetal human EECs from right and left ventricles with increasing concentrations of ET-1 induced a dose-dependent sustained increase of free cytosolic and nuclear Ca2+ levels with an EC50 near 10(-10) M and 10(-11) M, respectively. The sustained increase of cytosolic and nuclear Ca2+ by ET-1 in both EEC preparations was completely blocked by the calcium chelator ethylene glycol-bis (beta-aminoethylether)-N,N,N',N'-tetra-acetic acid (EGTA) but was insensitive to the L-type Ca2+ channel blocker, nifedipine. The effect of ET-1 was prevented by the ET(A)-receptor antagonist BQ 123. However the ET(B)-receptor antagonist BQ 788 had no effect. Our results suggest that ET-1 receptors are present in human EECs and that their stimulation induces sustained Ca2+ influx through ET(A)-receptor stimulation of a nifedipine-insensitive Ca2+ channel, probably the R-type Ca2+ channel. The presence of Ca2+-dependent responses to ET-1 supports a possible modulatory role of the endocardial endothelium in myocardial functions.

Presence of functional endothelin-1 receptors in nuclear membranes of human aortic vascular smooth muscle cells.

Our previous work showed that the nucleus plays a role in excitation-contraction coupling and that the channels and receptors could be present at the nuclear membrane. In the study reported here, the objective was to test the hypothesis that endothelin-1 (ET-1) receptors are functional at the level of the nuclear membranes and that their stimulation importantly regulates free nucleoplasmic Ca2+ level. Using a Fluo-3 Ca2+ measurement technique in human vascular smooth muscle cells (HVSMC), superfusion with increasing concentrations of extracellular ET-1 induced a dose-dependent sustained increase of free cytosolic ([Ca]c), nuclear ([Ca]n) Ca2+ and contraction with an EC50 near 3 x 10(-10) M. Like the extracellular ET-1, the cytosolic application of ET-1 using the perforated sarcolemma membrane technique, induced a dose-dependent increase of nuclear free calcium of HVSMC with an EC50 of 2 x 10(-11) M. These results strongly suggest that ET-1 receptors are functional at the level of the nuclear membranes. Furthermore, the sensitivity of ET-1 receptors at the nuclear membrane level seems to be higher than that of the receptors at the sarcolemma membrane. Finally, our results suggest that cytosolic ET-1 may play a role in preventing HVSMC nuclear calcium overload, thus protecting the cells from apoptosis.