Epigenomic Convergence of Neural-Immune Risk Factors in Neurodevelopmental Disorder Cortex.

Neurodevelopmental disorders (NDDs) affect 7-14% of all children in developed countries and are one of the leading causes of lifelong disability. Epigenetic modifications are poised at the interface between genes and environment and are predicted to reveal insight into NDD etiology. Whole-genome bisulfite sequencing was used to examine DNA cytosine methylation in 49 human cortex samples from 3 different NDDs (autism spectrum disorder, Rett syndrome, and Dup15q syndrome) and matched controls. Integration of methylation changes across NDDs with relevant genomic and genetic datasets revealed differentially methylated regions (DMRs) unique to each type of NDD but with shared regulatory functions in neurons and microglia. NDD DMRs were enriched within promoter regions and for transcription factor binding sites with identified methylation sensitivity. DMRs from all 3 disorders were enriched for ontologies related to nervous system development and genes with disrupted expression in brain from neurodevelopmental or neuropsychiatric disorders. Genes associated with NDD DMRs showed expression patterns indicating an important role for altered microglial function during brain development. These findings demonstrate an NDD epigenomic signature in human cortex that will aid in defining therapeutic targets and early biomarkers at the interface of genetic and environmental NDD risk factors.

X chromosome gene methylation in peripheral lymphocytes from monozygotic twins discordant for scleroderma.

Scleroderma (SSc) is a rare connective tissue disease characterized by fibrosis, microvasculopathy and autoimmune features. The role of genetics is limited in SSc, as suggested by similar concordance rates in monozygotic and dizygotic twin pairs, while environmental factors may act through epigenetic changes, as demonstrated for specific genes. Further, sex chromosome changes have been reported in SSc and may explain the female preponderance. In the present study we compared the methylation profile of all X chromosome genes in peripheral blood mononuclear cells from monozygotic twins discordant (n=7) and concordant (n=1) for SSc. Methylated DNA immunoprecipitations from each discordant twin pair were hybridized to a custom-designed array included 998 sites encompassing promoters of all X chromosome genes and randomly chosen autosomal genes. Biostatistical tools identified sites with an elevated probability to be consistently hypermethylated (n=18) or hypomethylated (n=25) in affected twins. Identified genes include transcription factors (ARX, HSFX1, ZBED1, ZNF41) and surface antigens (IL1RAPL2, PGRMC1), and pathway analysis suggests their involvement in cell proliferation (PGK1, SMS, UTP14A, SSR4), apoptosis (MTM1), inflammation (ARAF) and oxidative stress (ENOX2). In conclusion, we propose that X chromosome genes with different methylation profiles in monozygotic twin pairs may constitute candidates for SSc susceptibility.

Chromosome 15q11-13 duplication syndrome brain reveals epigenetic alterations in gene expression not predicted from copy number.

BACKGROUND: Chromosome 15q11-13 contains a cluster of imprinted genes essential for normal mammalian neurodevelopment. Deficiencies in paternal or maternal 15q11-13 alleles result in Prader-Willi or Angelman syndromes, respectively, and maternal duplications lead to a distinct condition that often includes autism. Overexpression of maternally expressed imprinted genes is predicted to cause 15q11-13-associated autism, but a link between gene dosage and expression has not been experimentally determined in brain. METHODS: Postmortem brain tissue was obtained from a male with 15q11-13 hexasomy and a female with 15q11-13 tetrasomy. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure 10 15q11-13 transcripts in maternal 15q11-13 duplication, Prader-Willi syndrome, and control brain samples. Southern blot, bisulfite sequencing and fluorescence in situ hybridisation were used to investigate epigenetic mechanisms of gene regulation. RESULTS: Gene expression and DNA methylation correlated with parental gene dosage in the male 15q11-13 duplication sample with severe cognitive impairment and seizures. Strikingly, the female with autism and milder Prader-Willi-like characteristics demonstrated unexpected deficiencies in the paternally expressed transcripts SNRPN, NDN, HBII85, and HBII52 and unchanged levels of maternally expressed UBE3A compared to controls. Paternal expression abnormalities in the female duplication sample were consistent with elevated DNA methylation of the 15q11-13 imprinting control region (ICR). Expression of non-imprinted 15q11-13 GABA receptor subunit genes was significantly reduced specifically in the female 15q11-13 duplication brain without detectable GABRB3 methylation differences. CONCLUSION: Our findings suggest that genetic copy number changes combined with additional genetic or environmental influences on epigenetic mechanisms impact outcome and clinical heterogeneity of 15q11-13 duplication syndromes.

Detection of low level HER-2/neu gene amplification in prostate cancer by fluorescence in situ hybridization.

PURPOSE: Although expression of the HER-2/neu oncogene has been correlated with tumor progression in prostate cancer, the biologic significance of detecting HER-2/neu gene amplification by fluorescence in situ hybridization (FISH) or evidence for protein overexpression by immunohistochemistry (IHC) remains unclear. In this study, we directly compared HER-2/neu FISH and IHC to determine which may be more predictive of the response to trastuzumab. PATIENTS AND METHODS: Forty patients with prostate cancer were analyzed for gene amplification by FISH performed with HER-2/neu and chromosome 17 (CEP 17) DNA probes (Vysis). Protein expression was examined by immunofluorescence and by IHC using the DAKO HercepTest antibody protocol and a monoclonal antibody to Her-2/neu on archival paraffin sections. The patients included 30 men with primary tumors that were treated with radical prostatectomy. Of these, 15 demonstrated subsequent disease progression within 3 years. Five patients with prostatic intraepithelial neoplasia were tested, as were five with metastatic disease whose samples were obtained before androgen ablation therapy. RESULTS: None of the 30 primary prostate cancer specimens showed overexpression for HER-2/neu by immunofluorescence or by IHC with the DAKO protocol. One sample showed 3+ membrane expression with the monoclonal antibody. In contrast, low copy number gene amplifications (3-8 HER-2/neu signals/nucleus) were detected in 16 of 30 samples (53%) by FISH. Most amplified cells were diploid for CEP 17, demonstrating that amplification was not due to total cell aneuploidy. FISH and IHC determined that prostatic intraepithelial neoplasia samples were normal. Four of five (80%) metastatic samples were amplified for HER-2/neu by FISH. Nearly 70% of metastatic cancer cells among all five specimens demonstrated aneuploidy. A single lymph node metastasis showed 3+ membrane staining by IHC (DAKO). CONCLUSIONS: In contrast to breast cancer, FISH detects HER-2/neu amplification in a substantial proportion of prostate cancers that do not overexpress HER-2/neuby IHC. Although the biologic significance of this finding is uncertain, it has implications for the direction of current and planned clinical trials of trastuzumab in advanced prostate cancer, including determination of patient eligibility.

Quantitative localization of heterogeneous methyl-CpG-binding protein 2 (MeCP2) expression phenotypes in normal and Rett syndrome brain by laser scanning cytometry.

Rett syndrome (RTT) is an X-linked, dominant neurodevelopmental disorder caused by mutations in MECP2, encoding the methyl-CpG-binding protein 2 (MeCP2). A major paradox in the pathogenesis of RTT is how mutations in ubiquitously transcribed MECP2 result in a phenotype specific to the central nervous system (CNS) during postnatal development. To address this question, we have used a novel approach for quantitating the level and distribution of wild-type and mutant MeCP2 in situ by immunofluorescence and laser scanning cytometry. Surprisingly, cellular heterogeneity in MeCP2 expression level was observed in normal brain with a subpopulation of cells exhibiting high expression (MeCP2(hi)) and the remainder exhibiting low expression (MeCP2(lo)). MeCP2 expression was significantly higher in CNS compared with non-CNS tissues of human and mouse by automated quantitation of MeCP2 on multiple tissue arrays. Quantitative localization of MeCP2 expression phenotypes in normal human brain showed a mosaic, but distinct, distribution pattern, with MeCP2(hi) neurons highest in layer IV of the cerebrum and MeCP2(lo )neurons highest in the granular layer of the cerebellum. In female RTT brains, MECP2 mutant-expressing cells were identified as cells negative for the MeCP2 C-terminal epitope. MECP2 mutant-expressing cells were randomly localized in Rett cerebrum and cerebellum and showed normal MeCP2 expression with N-terminal-specific anti-MeCP2. These results demonstrate a CNS-specific cellular phenotype of MeCP2 high expression and suggest that MECP2 mutations in RTT are only manifested in MeCP2(hi) cells. In addition, our results demonstrate the power of laser scanning cytometry in examining complex cellular phenotypes in disease pathogenesis.

Clonal maintenance of imprinted expression of SNRPN and IPW in normal lymphocytes: correlation with allele-specific methylation of SNRPN intron 1 but not intron 7.

DNA methylation is a heritable and reversible modification to CpG sites in the mammalian genome. Parental allele-specific methylation is hypothesized to be important in the establishment and maintenance of imprinted gene expression; however, dynamic changes in allele-specific patterns have been observed. The upstream regulatory region of the small nuclear riboprotein N gene (SNRPN) is an important imprinting control region (ICR) for establishing and maintaining the methylation imprint in the locus on 15q11-13 associated with Prader-Willi and Angelman syndromes (PWS). To compare directly the role of allele-specific methylation patterns and the maintenance of imprinted expression in the PWS region, clonal populations of normal T lymphocytes were cultured for 22-25 generations. A novel long-range semi-nested polymerase chain reaction (PCR) strategy was utilized in order to span two different methylation sites, and a polymorphism within SNRPN was used so that allele-specific methylation of both sites could be determined. Reverse transcription/PCR followed by polymorphism analysis was also performed in order to determine parental allele-specific transcription. Exclusive paternal expression at both SNRPN and IPW was maintained in all T cell clones and correlated with maternal methylation of the intron 1 NotI site. In contrast, biallelic methylation was observed in all clones at the previously described paternally methylated HpaII site in intron 7. These results demonstrate that the maintenance of paternal expression of SNRPN and IPW correlates with a strict clonal maintenance of allele-specific methylation at the CpG-dense 5' end of SNRPN. Differential maintenance of methylation sites within imprinted genes may depend on the density and chromatin organization of surrounding CpG sites.

Clonal heterogeneity at allelic methylation sites diagnostic for Prader-Willi and Angelman syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are developmental disorders resulting from the absence of the paternal or maternal contribution to the 15q11-13 region, respectively. Allele-specific methylation at D15S63 (PW71) has routinely been used as a diagnostic indicator of PWS and AS in DNA samples derived from peripheral blood. Extensive variation in allele-specific methylation patterns, however, has been observed at this site in different tissues, but the frequency or mechanism of this variation has remained uncharacterized. Herein, we have investigated the cellular basis of variation in methylation patterns at four sites of allelic methylation near SNRPN by using DNA samples derived from a panel of primary T lymphocyte clones. Interclonal variability was observed at three of these sites, including the diagnostic PW71 site. Changes in allele-specific methylation patterns occurred at a frequency of about one change in 50% of the cells every 22-25 doublings. In contrast, stable allele-specific methylation was observed in these clonal populations at exon 1 of SNRPN and the androgen receptor locus on the inactive X chromosome, suggesting that methylation at some CpG sites is more faithfully maintained than others. Clonal heterogeneity at PW71 was not an artifact of cell culture because the absence of allelic methylation was also observed in about 20% of the alleles in unstimulated peripheral blood. These results demonstrate that variation in allele-specific methylation at PW71 and other sites in the PWS/AS region appear to depend on the clonal complexity of the particular tissue and on the lack of strict maintenance of methylation within clones.

High-resolution analysis of DNA replication domain organization across an R/G-band boundary.

Establishing how mammalian chromosome replication is regulated and how groups of replication origins are organized into replication bands will significantly increase our understanding of chromosome organization. Replication time bands in mammalian chromosomes show overall congruency with structural R- and G-banding patterns as revealed by different chromosome banding techniques. Thus, chromosome bands reflect variations in the longitudinal structure and function of the chromosome, but little is known about the structural basis of the metaphase chromosome banding pattern. At the microscopic level, both structural R and G bands and replication bands occupy discrete domains along chromosomes, suggesting separation by distinct boundaries. The purpose of this study was to determine replication timing differences encompassing a boundary between differentially replicating chromosomal bands. Using competitive PCR on replicated DNA from flow-sorted cell cycle fractions, we have analyzed the replication timing of markers spanning roughly 5 Mb of human chromosome 13q14.3/q21.1. This is only the second report of high-resolution analysis of replication timing differences across an R/G-band boundary. In contrast to previous work, however, we find that band boundaries are defined by a gradient in replication timing rather than by a sharp boundary separating R and G bands into functionally distinct chromatin compartments. These findings indicate that topographical band boundaries are not defined by specific sequences or structures.

RAB22 and RAB163/mouse BRCA2: proteins that specifically interact with the RAD51 protein.

The human RAD51 protein is a homologue of the bacteria RecA and yeast RAD51 proteins that are involved in homologous recombination and DNA repair. RAD51 interacts with proteins involved in recombination and also with tumor suppressor proteins p53 and breast cancer susceptibility gene 1 (BRCA1). We have used the yeast two-hybrid system to clone murine cDNA sequences that encode two RAD51-associated molecules, RAB22 and RAB163. RAB163 encodes the C-terminal portion of mouse BRCA2, the homologue of the second breast cancer susceptibility gene protein in humans, demonstrating an in vitro association between RAD51 and BRCA2. RAB22 is a novel gene product that also interacts with RAD51 in vitro. To detect RAD51 interactions in vivo, we developed a transient nuclear focus assay that was used to demonstrate a complete colocalization of RAB22 with RAD51 in large nuclear foci.

Homologous association of oppositely imprinted chromosomal domains.

Human chromosome 15q11-q13 encompasses the Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) loci, which are subject to parental imprinting, a process that marks the parental origin of certain chromosomal subregions. A temporal and spatial association between maternal and paternal chromosomes 15 was observed in human T lymphocytes by three-dimensional fluorescence in situ hybridization. This association occurred specifically at the imprinted 15q11-q13 regions only during the late S phase of the cell cycle. Cells from PWS and AS patients were deficient in association, which suggests that normal imprinting involves mutual recognition and preferential association of maternal and paternal chromosomes 15.

Domain organization of allele-specific replication within the GABRB3 gene cluster requires a biparental 15q11-13 contribution.

Imprinting marks the parental origin of chromosomes, resulting in allele-specific changes in chromatin organization, transcription and replication. We report a 50-60 kb domain of allele-specific replication between the gamma-aminobutyric acid receptor subunit beta 3 (GABRB3) and alpha 5 (GABRA5) genes. Replication of this domain occurs in early S phase on the maternal chromosome 15 but is delayed until the end of S phase on the paternal homologue. In contrast, the genomic regions flanking this domain exhibit paternal earlier replication in mid to late S phase. Uniparental disomy or hemizygous deletion of chromosome 15 results in altered allele-specific replication kinetics compared with normals, suggesting that allele-specific replication within the GABRB3/A5 region may be regulated by reciprocal imprints on the maternal and paternal chromosomes.

T cell anergy.

T cell clonal anergy is a proposed mechanism of immunologic self tolerance in which T cells become functionally inactivated after previous stimulation. MHC class II-restricted antigen presentation by different cell types has been speculated to have a role in determining activation vs. anergy in responding T cells. Human T cells express MHC class II after activation and have been shown to present high concentrations of degraded peptide antigen to autologous T cells resulting in clonal anergy. In contrast to low antigen dose T cell clonal anergy, which occurs in the absence of costimulation, T cell anergy induced by human T cell presentation of antigen results in both primary proliferation and secondary unresponsiveness to high-dose antigenic stimulation. Although clonal anergy was previously thought to prevent autoreactive T cells from ever responding to self antigen presented without costimulation, we postulate that T cell presentation of antigen represents a "fail-safe" mechanism of immunologic self tolerance that would anergize clonally expanded autoreactive T cells when they are surrounded by a high extracellular concentration of degraded self antigen.

T-cell presentation of antigen requires cell-to-cell contact for proliferation and anergy induction. Differential MHC requirements for superantigen and autoantigen.

MHC class II+ human T-cell clones are able to simultaneously present and respond to peptide Ag and superantigen resulting in both proliferation and subsequent anergy. A major question remains as to whether a single T cell can present to itself or whether T-T cell interactions are required. We have employed a novel technique for inhibiting cell-to-cell contact that encapsulates individual T cells in agarose gel microdrops. Myelin basic protein-reactive individual CD4+ T-cell clones entrapped within these microdrops neither proliferated nor became anergized to either peptide Ag or Staphylococcal enterotoxin B (SEB), suggesting that cell-to-cell contact was required for T-cell presentation of Ag leading to proliferation and anergy. PMA treatment induced T-cell migration out of gel microdrops, restoring cell-to-cell contact and resulting in proliferation and anergy after T-cell coculture with peptide or superantigen. However, analysis of [Ca+2]i release revealed differences in T-cell responses to SEB versus peptide Ag. The addition of SEB, but not peptide Ag, induced a calcium flux in solitary T cells. Additionally, alpha HLA-DR mAb blocked peptide but not SEB-induced proliferation and anergy induction. Thus, SEB generated an early signal in solitary T cells that may not be a result of self stimulation via MHC class II. However, subsequent cell-to-cell contact was required for proliferation and anergy induction by SEB. These results indicate that peptide Ag requires a MHC class II-dependent cell-to-cell interaction for calcium flux, proliferation, and anergy induction, whereas SEB requires a MHC class II independent cell-to-cell interaction for proliferation and anergy induction after a TCR-generated calcium flux.

Biological activity of recombinant human myelin basic protein.

Using an inducible expression vector in Escherichia coli, we have expressed, purified, and biologically characterized recombinant human myelin basic protein (r-MBP). The recombinant protein binds in cation-exchange chromatography with similar affinity to purified, human MBP, and elutes as a single, 18.5-kDa species as judged by SDS-PAGE. The recombinant protein exhibits similar conformation to native MBP, as demonstrated by ELISA reactivity with a panel of six monoclonal antibodies directed against at least three different epitopes on human MBP. Additionally, recombinant MBP can trigger the proliferation of human T cell clones recognizing MBP, can induce experimental autoimmune encephalomyelitis (EAE) in the SJL mouse, and is capable of suppressing EAE following tolerization via oral administration. Most important, by using a novel method of purification, the recombinant protein preparation contains no detectable proteolytically derived fragments of MBP, a significant advantage over MBP purified from protease-rich central nervous system tissue. Purified recombinant MBP produced in E. coli will be useful as a biological reagent where highly purified protein devoid of degradation products is needed. Relevance to the study of antigen processing, interactions between MHC and the TCR, as well as for the investigation of antigen-driven immune tolerance are discussed.

Early signaling defects in human T cells anergized by T cell presentation of autoantigen.

Major histocompatibility complex class II-positive human T cell clones are nontraditional antigen-presenting cells (APCs) that are able to simultaneously present and respond to peptide or degraded antigen, but are unable to process intact protein. Although T cell presentation of peptide antigen resulted in a primary proliferative response, T cells that had been previously stimulated by T cells presenting antigen were completely unresponsive to antigen but not to interleukin 2 (IL-2). In contrast, peptide antigen presented by B cells or DR2+ L cell transfectants resulted in T cell activation and responsiveness to restimulation. The anergy induced by T cell presentation of peptide could not be prevented by the addition of either autologous or allogeneic B cells or B7+ DR2+ L cell transfectants, suggesting that the induction of anergy could occur in the presence of costimulation. T cell anergy was induced within 24 h of T cell presentation of antigen and was long lasting. Anergized T cells expressed normal levels of T cell receptor/CD3 but were defective in their ability to release [Ca2+]i to both alpha CD3 and APCs. Moreover, anergized T cells did not proliferate to alpha CD2 monoclonal antibodies or alpha CD3 plus phorbol myristate acetate (PMA), nor did they synthesize IL-2, IL-4, or interferon gamma mRNA in response to either peptide or peptide plus PMA. In contrast, ionomycin plus PMA induced both normal proliferative responses and synthesis of cytokine mRNA, suggesting that the signaling defect in anergized cells occurs before protein kinase C activation and [Ca2+]i release.

The coexpression of CD45RA and CD45RO isoforms on T cells during the S/G2/M stages of cell cycle.

The tyrosine phosphatase CD45 is alternatively spliced to generate isoforms of different molecular weights (180-220 kDa) which are differentially expressed on hematopoietic cells. Monoclonal antibodies reacting with either the 180-kDa (UCHL-1, CD45RO) or the 200- to 220-kDa (2H4, CD45RA) isoform have been used to subdivide T cell populations based on their expression of one or the other of these two epitopes. CD45RA T cells have "naive" characteristics of unresponsiveness to recall antigens and prominence in cord blood, while CD45RO T cells are considered "memory" T cells because they proliferate to recall antigens and increase following PHA activation of cord blood. However, we have recently demonstrated the expression of the CD45RA isoform on a subpopulation of CD45RO+ T cell clones, suggesting that CD45RA is not a universal marker for naive T cells. Using propidium iodide staining of the DNA to determine cell cycle stage, we now show that CD45RA expression is significantly higher on T cell clones during the S, G2, and M stages of cell cycle when compared to CD45RA expression on cells in Go and G1. Furthermore, CD45RA expression on cells undergoing mitosis is not limited to long-term activated T cell clones, as uncultured peripheral blood T cells in the S/G2/M phase express significantly more CD45RA. The percentage of T cells coexpressing CD45RA and CD45RO also increases following PHA activation, indicating that T cells in the process of division express both isoforms. These results suggest a potential role of the CD45RA isoform during the stages of cell cycle leading to mitosis.

Presentation of autoantigen by human T cells.

Activated human T cells express MHC class II and have been shown to present foreign Ag to autologous T cells. We now demonstrate that MHC class II+ T cell clones can present myelin basic protein (MBP) peptide autoantigen in the absence of traditional APC to autologous MBP reactive T cell clones. MBP peptide-pulsed T cell clones specifically stimulated autologous MBP-reactive T cell clones to flux calcium and proliferate. Activation responses were peptide epitope specific and blocked by mAb to MHC class II, indicating a TCR-mediated response. In addition, mAb to the adhesion molecules LFA-3, CD2, LFA-1, CD29, and to the tyrosine phosphatase CD45 also inhibited proliferation, indicating the involvement of T to T cell interactions. In contrast to peptide Ag, T cell clones did not respond to autologous T cells pulsed with HPLC-purified MBP, suggesting that T cells are unable to process whole MBP. However, batch-purified MBP Ag preparations containing lower m.w. breakdown products were presented by T cells, indicating that naturally occurring breakdown products of autoantigens could be presented by activated T cells in vivo. These results raise the possibility that T cell presentation of autoantigen at inflammatory sites may be important in regulation of immune responses to self Ag.