Stephen J. Glatt

Analysis of protein-coding genetic variation in 60,706 humans

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human ‘knockout’ variants in protein-coding genes.

Hypermethylation of the reelin (RELN) promoter in the brain of schizophrenic patients: A preliminary report

DNA methylation changes could provide a mechanism for DNA plasticity and dynamism for short‐term adaptation, enabling a type of cell memory to register cellular history under different environmental conditions. Some environmental insults may also result in pathological methylation with corresponding alteration of gene expression patterns. Evidence from several studies has suggested that in schizophrenia and bipolar disorder, mRNA of the reelin gene (RELN), which encodes a protein necessary for neuronal migration, axonal branching, synaptogenesis, and cell signaling, is severely reduced in post‐mortem brains. Therefore, we investigated the methylation status of the RELN promoter region in schizophrenic patients and normal controls as a potential mechanism for down regulation of its expression. Ten post‐mortem frontal lobe brain samples from male schizophrenic patients and normal controls were obtained from the Harvard Brain Tissue Resources Center. DNA was extracted using a standard phenol–chloroform DNA extraction protocol. To evaluate differences between patients and controls, we applied methylation specific PCR (MSP) using primers localized to CpG islands flanking a potential cyclic AMP response element (CRE) and a stimulating protein‐1 (SP1) binding site located in the promoter region. For each sample, DNA extraction, bisulfite treatment, and MSP were independently repeated at least four times to accurately determine the methylation status of the target region. Forty‐three PCR trials were performed on the test and control samples. MSP analysis of the RELN promoter revealed an unmethylated signal in all reactions (43 of 43) using DNA from the frontal brain tissue, derived from either the schizophrenic patients or normal controls indicating that this region of the RELN promoter is predominantly unmethylated. However, we observed a distinct methylated signal in 73% of the trials (16 of 22) in schizophrenic patients compared with 24% (5 of 21) of controls. Thus, the hypermethylation of the CpG islands flanking a CRE and SP1 binding site observed at a significantly higher level (t = −5.07, P = 0.001) may provide a mechanism for the decreased RELN expression, frequently observed in post‐mortem brains of schizophrenic patients. We also found an inverse relationship between the level of DNA methylation using MSP analysis and the expression of the RELN gene using semi‐quantitative RT‐PCR. Despite the small sample size, these studies indicate that promoter hypermethylation of the RELN gene could be a significant contributor in effecting epigenetic alterations and provides a molecular basis for the RELN gene hypoactivity in schizophrenia. Further studies with a larger sample set would be required to validate these preliminary observations.

Meta-analysis of the association between two polymorphisms in the serotonin transporter gene and affective disorders

Family, twin, and adoption studies show that psychiatric diseases including bipolar disorder (BP) and unipolar disorder (UP) have a substantial genetic component. For these illnesses, both positive and negative associations have been reported for two polymorphisms located in the serotonin transporter gene (5‐HTT) on chromosome 17: a 17‐base‐pair (bp) variable‐number tandem‐repeat (VNTR) in intron 2 and a 44‐bp insertion/deletion in the promoter region. Thus, associations between these 5‐HTT polymorphisms and affective disorders remain unclear. The present work investigates these potential associations in meta‐analyzes that maximize the power to find associations between each disease and the two 5‐HTT polymorphisms. We applied meta‐analysis techniques to case‐control studies of two 5‐HTT polymorphisms and two affective disorders (BP and UP), resulting in four meta‐analyzes. For each polymorphism, we assessed the evidence for allelic associations, heterogeneity among studies, the influence of individual studies, and the potential for publication bias. The short allele(s) of the 44‐bp insertion/deletion polymorphism showed a significant association for BP (odds ratio (OR) = 1.13, P = 0.001) but not UP. For the 17‐bp VNTR, an increase in the number of tandem repeats had no significant association with any of the disorders. The small but significant effects of the 44‐bp insertion/deletion polymorphism for BP is consistent with being one of many genes that contributes to the multi‐factorial nature of these psychiatric disorders.

Comparative gene expression analysis of blood and brain provides concurrent validation of SELENBP1 up-regulation in schizophrenia

Microarray techniques hold great promise for identifying risk factors for schizophrenia (SZ) but have not yet generated widely reproducible results due to methodological differences between studies and the high risk of type I inferential errors. Here we established a protocol for conservative analysis and interpretation of gene expression data from the dorsolateral prefrontal cortex of SZ patients using statistical and bioinformatic methods that limit false positives. We also compared brain gene expression profiles with those from peripheral blood cells of a separate sample of SZ patients to identify disease-associated genes that generalize across tissues and populations and further substantiate the use of gene expression profiling of blood for detecting valid SZ biomarkers. Implementing this systematic approach, we: (i) discovered 177 putative SZ risk genes in brain, 28 of which map to linked chromosomal loci; (ii) delineated six biological processes and 12 molecular functions that may be particularly disrupted in the illness; (iii) identified 123 putative SZ biomarkers in blood, 6 of which (BTG1, GSK3A, HLA-DRB1, HNRPA3, SELENBP1, and SFRS1) had corresponding differential expression in brain; (iv) verified the differential expression of the strongest candidate SZ biomarker (SELENBP1) in blood; and (v) demonstrated neuronal and glial expression of SELENBP1 protein in brain. The continued application of this approach in other brain regions and populations should facilitate the discovery of highly reliable and reproducible candidate risk genes and biomarkers for SZ. The identification of valid peripheral biomarkers for SZ may ultimately facilitate early identification, intervention, and prevention efforts as well.

A comparison of the efficacy of medications for adult attention-deficit/hyperactivity disorder using meta-analysis of effect sizes.

OBJECTIVES Medications used to treat attention-deficit/hyperactivity disorder (ADHD) in adults have been well researched, but comparisons among drugs are hindered by the absence of direct comparative trials. Our objectives were to (1) estimate the effect size of the medications used to treat adult ADHD, (2) determine if differences in the designs of studies confound comparisons of medication efficacy, (3) quantify the evidence for differences in effect sizes among medications, and (4) see if features of study design influence estimates of efficacy. DATA SOURCES The following search engines were used: PubMed, Ovid, ERIC, CINAHL, MEDLINE, PREMEDLINE, the Cochrane database, e-psyche, and Social Sciences Abstracts. Presentations from the American Psychiatric Association and American Academy of Child and Adolescent Psychiatry meetings were reviewed. STUDY SELECTION A literature search was conducted to identify double-blind, placebo-controlled studies of ADHD in adults published in English after 1979. Only trials that used DSM-III, -III-R, or -IV ADHD criteria and followed subjects for > or = 2 weeks were selected. DATA EXTRACTION Meta-analysis regression assessed the influence of medication type and study design features on medication effects. RESULTS Nineteen trials met criteria and were included in this meta-analysis. These trials studied 13 drugs using 18 different outcome measures of hyperactive, inattentive, or impulsive behavior. After trials were stratified on the class of drug studied (short-acting stimulant vs long-acting stimulant vs nonstimulant), significant differences in effect size were observed between stimulant and nonstimulant medications (P = .006 and P = .0001, respectively, for short- and long-acting stimulants vs nonstimulants), but the effect for short-acting stimulants was not significant after correcting for study design features. The effect sizes for each drug class were similar in magnitude to what we previously reported for medication treatment studies of children with ADHD. We found significant heterogeneity of effect sizes for short-acting stimulants (P < .001) but not for other medication groups. CONCLUSIONS Although both stimulant and nonstimulant medications are effective for treating ADHD in adults, stimulant medications show greater efficacy for the short durations of treatment characteristic of placebo-controlled studies. We found no significant differences between short- and long-acting stimulant medications. Study design features vary widely among studies and can confound indirect comparisons unless addressed statistically as we have done in this study.

Psychopharmacology and aggression. I: A meta-analysis of stimulant effects on overt/covert aggression-related behaviors in ADHD.

OBJECTIVE To determine by meta-analysis the effect size for stimulants on overt and covert aggression-related behaviors in children with attention-deficit/hyperactivity disorder (ADHD), separately from stimulant effects on the core symptoms of ADHD. METHOD A review of the literature from 1970 to 2001 revealed 28 studies meeting inclusion/exclusion criteria for meta-analysis. These studies yielded 28 independent effects of overt aggression and 7 independent effects of covert aggression. RESULTS The overall weighted mean effect size was 0.84 for overt and 0.69 for covert aggression related behaviors in ADHD. Comorbid conduct disorder is associated with diminishing stimulant effect size for overt aggression. CONCLUSION Stimulant effects for aggression-related behaviors in ADHD have effect sizes similar to those for the core symptoms of ADHD.

Assessing the validity of blood-based gene expression profiles for the classification of schizophrenia and bipolar disorder: a preliminary report.

Recent advances have facilitated the use of blood‐derived RNA to conduct genomic analyses of human diseases. This emerging technology represents a rigorous and convenient alternative to traditional tissue biopsy‐derived RNA, as it allows for larger sample sizes, better standardization of technical procedures, and the ability to non‐invasively profile human subjects. In the present pilot study, we have collected RNA from blood of patients diagnosed with schizophrenia or bipolar disorder (BPD), as well as normal control subjects. Using microarray analysis, we found that each disease state exhibited a unique expressed genome signature, allowing us to discriminate between the schizophrenia, BPD, and control groups. In addition, we validated changes in several potential biomarker genes for schizophrenia and BPD by RT‐PCR, and some of these were found to code to chromosomal loci previously linked to schizophrenia. Linear and non‐linear combinations of eight putative biomarker genes (APOBEC3B, ADSS, ATM, CLC, CTBP1, DATF1, CXCL1, and S100A9) were able to discriminate between schizophrenia, BPD, and control samples, with an overall accuracy of 95%–97% as indicated by receiver operating characteristic (ROC) curve analysis. We therefore propose that blood cell‐derived RNA may have significant value for performing diagnostic functions and identifying disease biomarkers in schizophrenia and BPD.

Methylomics in psychiatry: Modulation of gene-environment interactions may be through DNA methylation.

Fine‐tuning of neuronal connections during development is regulated through environmental interactions. Some fine‐tuning occurs through changes in gene expression and/or epigenetic gene‐specific DNA methylation states. DNA methylation occurs by transfer of a methyl group from S‐adenosyl methionine to cytosine residues in the dinucleotide sequence CpG. Although CpG sequences spread throughout the genome are usually heavily methylated, those occurring in CpG islands in the promoter regions of genes are less methylated. In most cases, the extent of DNA methylation correlates with the extent of gene inactivation. Other known epigenetic mechanisms include histone deacetylation and chromatin remodeling, RNA inhibition, RNA modification, and DNA rearrangement. Exposure memory expressed as epigenetic DNA modifications allows genomic plasticity and short‐term adaptation of each generation to their environment. Environmental factors that affect DNA methylation include diet, proteins, drugs, and hormones. Induced methylation changes may produce altered gene response upon subsequent hormonal stimulation. The gene‐specific DNA methylation state may be preserved upon transmission through mitosis and meiosis. An increasing amount of data implicates a role for DNA methylation in multi‐factorial psychiatric disorders. For example, l‐methionine treatment can exacerbate psychosis; while valproate, a drug producing hypomethylated DNA, reduces such symptoms. Hypermethylation of the promoter region of the RELN gene correlates with reduced gene expression. This genes protein Reelin, which is necessary for neuronal migration and synaptogenesis, is reduced in schizophrenia and bipolar disorder, suggesting hypermethylation of the promoter region in these disorders. Some evidence implicates methylation of the promoter regions of the DRD2 and HTR2A genes in schizophrenia and mood disorders as well. DNA methylation usually increases with age, although hypomethylation of the promoter region of the amyloid A4 precursor gene during aging may play a role in Alzheimers disease. More studies are needed to define the role of methylomics and other epigenetic phenomena in the nervous system.

Subsensitivity to dopaminergic drugs in periadolescent rats: a behavioral and neurochemical analysis

It has been reported that post-natal day (PD) 30-40 rats respond differently to the behavioral effects of dopaminergic drugs when compared to younger or older rats. In this study, the behavioral effects of amphetamine (AMPH) on motor behavior and the effects of dopaminergic drugs on striatal acetylcholine (ACh) release were evaluated in periadolescent (PD35) and adult rats. AMPH increased dopamine (DA)-mediated motor behaviors (locomotor activity and stereotypy) in periadolescent and adult rats; however, these responses were of a lesser magnitude in periadolescent rats. In adult rats, cocaine and nomifensine inhibited ACh overflow in a dose-dependent manner. In periadolescent rats, ACh overflow was maximally inhibited at a lower drug concentration (5 microM) than in adult rats (10 microM) signifying increased sensitivity in these rats. Apomorphine inhibited ACh overflow in a dose-dependent fashion in slices from adult rats. In contrast, apomorphine did not consistently inhibit ACh overflow in striatal slices prepared from periadolescent rats. Collectively, the results of this study demonstrate behavioral subsensitivity to AMPH in periadolescent rats. Examination of the effects of DA reuptake blockers on DA modulation of striatal cholinergic neurons failed to reveal a corresponding subsensitivity. In fact, ACh release was more sensitive to DA reuptake blockers in periadolescent rats. This latter finding suggests that undisclosed factors override dopaminergic modulation of striatal neurons in the mediation of behavior in periadolescent rats. We propose that during periadolescence, DA transmission is transiently elevated. This results in post-synaptic supersensitivity of cholinergic receptors and consequently induces behavioral subsensitivity when challenged with dopaminergic drugs. Increased cholinergic tone may mediate behavioral subsensitivity despite drug-induced elevations in DA.

Meta-analysis of association between the T102C polymorphism of the 5HT2a receptor gene and schizophrenia

A meta-analysis of whole-genome linkage scans confirmed linkage between schizophrenia and markers on the long arm of chromosome 13. The gene HTR2A, which codes for the 5HT2a receptor, is located in this area. The T102C single nucleotide polymorphism of HTR2A has been the subject of much research. The production of the C-allele form of HTR2A is significantly less than that of the T-allele form in normal controls and schizophrenic patients. Although the association of schizophrenia with the C allele of HTR2A was confirmed by a meta-analysis 5 years ago, there was a continuous debate because negative findings were also considerable, which may have been due to ethnic differences in association. We performed another meta-analysis, since the number of available studies of this association has recently doubled. In the meta-analysis of 31 case-control association studies, we found a significant association between the C allele of the T102C polymorphism and schizophrenia, which was more pronounced in European samples than in the entire sample. We found significant heterogeneity in the allele-wise analysis (C vs. T) and homozygous genotype-wise analysis (CC vs. TT), both of which were at least partially explained by differences between samples from Asian and European countries. In East Asian countries, there was not a significant association with the C allele or CC homozygosity, indicating strong genetic differences and noncombinability of data between European and East Asian populations. Interestingly, the frequency of the T allele was much higher in East Asian patients and controls (59.5% and 57.5%, respectively) than in European patients and controls (40% and 43.5%, respectively). In five family-based association studies, we did not find significant evidence for association of the C allele with schizophrenia; yet, the pooled OR was 1.3 (95% CI=0.9-1.8, z=1.47, p=0.14), which is consistent with the results of the case-control studies. The effects of other genes, environmental effects on DNA methylation, or different methods of classification may be the causes for such heterogeneity, but more study in this area is needed.