Genomewide DNA methylation analysis in combat veterans reveals a novel locus for PTSD.

OBJECTIVE: Epigenetic modifications such as DNA methylation may play a key role in the aetiology and serve as biomarkers for post-traumatic stress disorder (PTSD). We performed a genomewide analysis to identify genes whose DNA methylation levels are associated with PTSD. METHOD: A total of 211 individuals comprising Australian male Vietnam War veterans (n = 96) and males from a general population belonging to the Grady Trauma Project (n = 115) were included. Genomewide DNA methylation was performed from peripheral blood using the Illumina arrays. Data analysis was performed using generalized linear regression models. RESULTS: Differential DNA methylation of 17 previously reported PTSD candidate genes was associated with PTSD symptom severity. Genomewide analyses revealed CpG sites spanning BRSK1, LCN8, NFG and DOCK2 genes were associated with PTSD symptom severity. We replicated the findings of DOCK2 in an independent cohort. Pathway analysis revealed that among the associated genes, genes within actin cytoskeleton and focal adhesion molecular pathways were enriched. CONCLUSION: These data highlight the role of DNA methylation as biomarkers of PTSD. The results support the role of previous candidates and uncover novel genes associated with PTSD, such as DOCK2. This study contributes to our understanding of the biological underpinnings of PTSD.

Brain-specific epigenetic markers of schizophrenia.

Epigenetics plays a crucial role in schizophrenia susceptibility. In a previous study, we identified over 4500 differentially methylated sites in prefrontal cortex (PFC) samples from schizophrenia patients. We believe this was the first genome-wide methylation study performed on human brain tissue using the Illumina Infinium HumanMethylation450 Bead Chip. To understand the biological significance of these results, we sought to identify a smaller number of differentially methylated regions (DMRs) of more functional relevance compared with individual differentially methylated sites. Since our schizophrenia whole genome methylation study was performed, another study analysing two separate data sets of post-mortem tissue in the PFC from schizophrenia patients has been published. We analysed all three data sets using the bumphunter function found in the Bioconductor package minfi to identify regions that are consistently differentially methylated across distinct cohorts. We identified seven regions that are consistently differentially methylated in schizophrenia, despite considerable heterogeneity in the methylation profiles of patients with schizophrenia. The regions were near CERS3, DPPA5, PRDM9, DDX43, REC8, LY6G5C and a region on chromosome 10. Of particular interest is PRDM9 which encodes a histone methyltransferase that is essential for meiotic recombination and is known to tag genes for epigenetic transcriptional activation. These seven DMRs are likely to be key epigenetic factors in the aetiology of schizophrenia and normal brain neurodevelopment.

Dysbindin (DTNBP1) variants are associated with hallucinations in schizophrenia.

BACKGROUND: Dystrobrevin binding protein 1 (DTNBP1) is a schizophrenia susceptibility gene involved with neurotransmission regulation (especially dopamine and glutamate) and neurodevelopment. The gene is known to be associated with cognitive deficit phenotypes within schizophrenia. In our previous studies, DTNBP1 was found associated not only with schizophrenia but with other psychiatric disorders including psychotic depression, post-traumatic stress disorder, nicotine dependence and opiate dependence. These findings suggest that DNTBP1 may be involved in pathways that lead to multiple psychiatric phenotypes. In this study, we explored the association between DTNBP1 SNPs (single nucleotide polymorphisms) and multiple psychiatric phenotypes included in the Diagnostic Interview of Psychosis (DIP). METHODS: Five DTNBP1 SNPs, rs17470454, rs1997679, rs4236167, rs9370822 and rs9370823, were genotyped in 235 schizophrenia subjects screened for various phenotypes in the domains of depression, mania, hallucinations, delusions, subjective thought disorder, behaviour and affect, and speech disorder. SNP-phenotype association was determined with ANOVA under general, dominant/recessive and over-dominance models. RESULTS: Post hoc tests determined that SNP rs1997679 was associated with visual hallucination; SNP rs4236167 was associated with general auditory hallucination as well as specific features including non-verbal, abusive and third-person form auditory hallucinations; and SNP rs9370822 was associated with visual and olfactory hallucinations. SNPs that survived correction for multiple testing were rs4236167 for third-person and abusive form auditory hallucinations; and rs9370822 for olfactory hallucinations. CONCLUSION: These data suggest that DTNBP1 is likely to play a role in development of auditory related, visual and olfactory hallucinations which is consistent with evidence of DTNBP1 activity in the auditory processing regions, in visual processing and in the regulation of glutamate and dopamine activity.

Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients.

Recent studies suggest that genetic and environmental factors do not account for all the schizophrenia risk, and epigenetics also has a role in disease susceptibility. DNA methylation is a heritable epigenetic modification that can regulate gene expression. Genome-wide DNA methylation analysis was performed on post-mortem human brain tissue from 24 patients with schizophrenia and 24 unaffected controls. DNA methylation was assessed at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 Bead Chip. After adjusting for age and post-mortem interval, 4641 probes corresponding to 2929 unique genes were found to be differentially methylated. Of those genes, 1291 were located in a CpG island and 817 were in a promoter region. These include NOS1, AKT1, DTNBP1, DNMT1, PPP3CC and SOX10, which have previously been associated with schizophrenia. More than 100 of these genes overlap with a previous DNA methylation study of peripheral blood from schizophrenia patients in which 27,000 CpG sites were analysed. Unsupervised clustering analysis of the top 3000 most variable probes revealed two distinct groups with significantly more people with schizophrenia in cluster one compared with controls (P=1.74 × 10(-4)). The first cluster composed of 88% of patients with schizophrenia and only 12% controls, whereas the second cluster composed of 27% of patients with schizophrenia and 73% controls. These results strongly suggest that differential DNA methylation is important in schizophrenia etiology and add support for the use of DNA methylation profiles as a future prognostic indicator of schizophrenia.

DRD2/ANKK1 Taq1A (rs 1800497 C>T) genotypes are associated with susceptibility to second generation antipsychotic-induced akathisia.

Although the advent of atypical, second-generation antipsychotics (SGAs) has resulted in reduced likelihood of akathisia, this adverse effect remains a problem. It is known that extrapyramidal adverse effects are associated with increased drug occupancy of the dopamine 2 receptors (DRD2). The A1 allele of the DRD2/ANKK1, rs1800497, is associated with decreased striatal DRD2 density. The aim of this study was to identify whether the A1(T) allele of DRD2/ANKK1 was associated with akathisia (as measured by Barnes Akathisia Rating Scale) in a clinical sample of 234 patients who were treated with antipsychotic drugs. Definite akathisia (a score ≥ 2 in the global clinical assessment of akathisia) was significantly less common in subjects who were prescribed SGAs (16.8%) than those prescribed FGAs (47.6%), p < 0.0001. Overall, 24.1% of A1+ patients (A1A2/A1A1) who were treated with SGAs had akathisia, compared to 10.8% of A1- (thus, A2A2) patients. A1+ patients who were administered SGAs also had higher global clinical assessment of akathisia scores than the A1- subjects (p = 0.01). SGAs maintained their advantage over FGAs regarding akathisia, even in A1+ patients who were treated with SGAs. These results strongly suggested that A1+ variants of the DRD2/ANKK1 Taq1A allele do confer an associated risk for akathisia in patients who were treated with SGAs, and these variants may explain inconsistencies found across prior studies, when comparing FGAs and SGAs.

HapMap tag-SNP analysis confirms a role for COMT in schizophrenia risk and reveals a novel association.

Catechol-O-methyl transferase (COMT) encodes an enzyme involved in the metabolism of dopamine and maps to a commonly deleted region that increases schizophrenia risk. A non-synonymous polymorphism (rs4680) in COMT has been previously found to be associated with schizophrenia and results in altered activity levels of COMT. Using a haplotype block-based gene-tagging approach we conducted an association study of seven COMT single nucleotide polymorphisms (SNPs) in 160 patients with a DSM-IV diagnosis of schizophrenia and 250 controls in an Australian population. Two polymorphisms including rs4680 and rs165774 were found to be significantly associated with schizophrenia. The rs4680 results in a Val/Met substitution but the strongest association was shown by the novel SNP, rs165774, which may still be functional even though it is located in intron five. Individuals with schizophrenia were more than twice as likely to carry the GG genotype compared to the AA genotype for both the rs165774 and rs4680 SNPs. This association was slightly improved when males were analysed separately possibly indicating a degree of sexual dimorphism. Our results confirm that COMT is a good candidate for schizophrenia risk, by replicating the association with rs4680 and identifying a novel SNP association.

Analysis of HapMap tag-SNPs in dysbindin (DTNBP1) reveals evidence of consistent association with schizophrenia.

Dystrobrevin binding protein 1 (DTNBP1), or dysbindin, is thought to be critical in regulating the glutamatergic system. While the dopamine pathway is known to be important in the aetiology of schizophrenia, it seems likely that glutamatergic dysfunction can lead to the development of schizophrenia. DTNBP1 is widely expressed in brain, levels are reduced in brains of schizophrenia patients and a DTNBP1 polymorphism has been associated with reduced brain expression. Despite numerous genetic studies no DTNBP1 polymorphism has been strongly implicated in schizophrenia aetiology. Using a haplotype block-based gene-tagging approach we genotyped 13 SNPs in DTNBP1 to investigate possible associations with DTNBP1 and schizophrenia. Four polymorphisms were found to be significantly associated with schizophrenia. The strongest association was found with an A/C SNP in intron 7 (rs9370822). Homozygotes for the C allele of rs9370822 were more than two and a half times as likely to have schizophrenia compared to controls. The other polymorphisms showed much weaker association and are less likely to be biologically significant. These results suggest that DTNBP1 is a good candidate for schizophrenia risk and rs9370822 is either functionally important or in disequilibrium with a functional SNP, although our observations should be viewed with caution until they are independently replicated.

Interrogation of multimeric DNA amplification products by competitive primer extension using bst DNA polymerase (large fragment).

Linear dsDNA composed of tandem repeats may be exponentially amplified by the strongly strand-displacing Bst DNA polymerase (large fragment) and two primers specific for opposite strands. When the repetitive DNA is derivedfrom rolling circle replication of a circular template, the reaction is termed cascade rolling circle amplification (CRCA). We have developed a variant of CRCA in which one primer is attached to the surface of a microwell and the other is labeled, thus enabling detection of amplified material using an ELISA-like protocol. The circular template is derived by annealing and ligation of a padlock on target DNA. It was found that there was good correlation between the synthesis of amplified material and signal. The specificity of the reaction with respect to single-nucleotide polymorphisms was investigated, and it was found that Bst DNA polymerase is prone to extension from primers with mismatched 3' ends. Reliable single nucleotide specificity was only obtained when pre-synthesized amplified material was interrogated by competitive primer extension.

Solid-phase amplification for detection of C282y and H63D hemochromatosis (HFE) gene mutations.

BACKGROUND: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples. METHODS: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. RESULTS: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results. CONCLUSIONS: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.

Solid-phase amplification and detection: a single-tube diagnostic assay for infectious agents.

BACKGROUND: We report the development of an enzyme-linked immunosorbent assay-like single-tube assay for the detection of infectious agents in a microtiter tray format. METHODS AND RESULTS: The method, sequential nucleic acid amplification and capture (SNAAC), combines amplification with hybridization of the product to a surface/matrix-bound oligonucleotide probe. After amplification of the target sequence using species-specific primers, one of which contains a detection tag such as fluorescein or biotin, a denaturation and hybridization cycle is performed. This allows capture by an oligonucleotide that is covalently bound to the surface of a microtiter tray well or other support. After washing to remove unincorporated solution-phase oligonucleotide bearing the detection tag, the level of captured product is determined through a colorimetric reaction using an automated plate reader. We show the value and utility of the SNAAC detection method using cloned sequences of the important human respiratory pathogen Chlamydia pneumoniae. CONCLUSIONS: SNAAC is a simple, rapid, and inexpensive method for the detection of low levels of infectious agents that is readily adaptable to current clinical laboratory equipment, thus avoiding the need to develop or purchase new instrumentation.

The factor V HR2 haplotype: prevalence and association of the A4070G and A6755G polymorphisms.

Recently, a polymorphism was identified in exon 25 of the factor V gene that is possibly a functional candidate for the HR2 haplotype. This haplotype is characterized by a single base substitution named R2 (A4070G) in the B domain of the protein. A mutation (A6755G; 2194Asp-->Gly) located near the C terminus has been hypothesized to influence protein folding and glycosylation, and might be responsible for the shift in factor V isoform (FV1 / FV2) ratio. This study investigated the prevalence of these two factor V HR2 haplotype polymorphisms in a cohort of normal blood donors, patients with osteoarthritis and women with complications during pregnancy, and in families of factor V Leiden individuals. A high allele frequency for the two polymorphisms was found in the blood donor group (6.2% R2, 5.6% A6755G). No significant difference in allele frequency was observed in the clinical groups (obstetric complications and osteoarthritis, 4.1-4.9% for the two polymorphisms) when compared with that of healthy blood donors. We confirm that the factor V A6755G polymorphism shows strong linkage to the R2 allele, although it is not exclusively inherited with the exon 13 A4070G variant and can occur independently.

Importance of the glycosylation and polyadenylation variants in metachromatic leukodystrophy pseudodeficiency phenotype.

Metachromatic leukodystrophy (MLD) is an inborn error of myelin metabolism caused by a deficiency of the lysosomal hydrolase, arylsulfatase A (ASA). About 1% of the normal population have ASA activity levels approximating those of MLD patients. This non-pathogenic reduction in ASA activity is caused by homozygosity for the ASA pseudodeficiency allele (ASA-PD). Although this allele contains two sequence alterations, a polyadenylation defect and an amino acid substitution (N350S), the reduction in ASA activity previously has been attributed to the polyadenylation defect which reduces the amount of ASA mRNA and hence ASA protein by approximately 90%. The identification of MLD patients who are homozygous for the ASA-PD allele has brought about the need to re-evaluate the allele in light of the possible role that it may play in the development and progression of disease. Ribonuclease protection assay analysis of ASA mRNA transcripts and an investigation into the activity and lysosomal localization of protein expressed by an ASA expression construct containing the N350S variant indicated that both the N350S and polyadenylation defects play a role in biochemically defining the ASA-PD phenotype. The combined effect of the reduction in ASA mRNA due to the polyadenylation defect and the lowering of ASA activity and aberrant targeting of the expressed N350S ASA protein to the lysosome is estimated to reduce ASA activity in pseudodeficiency homozygotes to approximately 8% of normal.

Altered responses of human macrophages to lipopolysaccharide by hydroperoxy eicosatetraenoic acid, hydroxy eicosatetraenoic acid, and arachidonic acid. Inhibition of tumor necrosis factor production.

The regulation of allergic and autoimmune inflammatory reactions by polyunsaturated fatty acids and their metabolic products (eicosanoids) continues to be of major interest. Our data demonstrate that arachidonic acid 5,8,11,14-eicosatetraenoic acid (20:4n-6) and its hydroxylated derivatives 15(s)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) and 15(s)-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) regulate agonist-induced tumor necrosis factor alpha (TNF) production, a cytokine that plays a role in inflammatory diseases. Although 20:4n-6 and 15-HETE caused a reduction in production of TNF in mononuclear leukocytes stimulated with phytohaemagglutinin, pokeweed mitogen, concanavalin A, and Staphylococcus aureus, 15-HPETE was far more active. 15-HPETE was also found to dramatically depress the ability of bacterial lipopolysaccharide to induce TNF production in monocytes and the monocytic cell line Mono Mac 6. These fatty acids depressed the expression of TNF mRNA in Mono Mac 6 cells stimulated with LPS; 15-HPETE was fivefold more active than 20:4n-6 and 15-HETE. While 15-HPETE treatment neither affected LPS binding to Mono Mac 6 cells nor caused a decrease in CD14 expression, the fatty acid significantly reduced the LPS-induced translocation of PKC (translocation of alpha, betaI, betaII, and epsilon isozymes), suggesting that 15-HPETE acts by abrogating the early signal transduction events. The findings identify another molecule that could form the basis for development of antiinflammatory pharmaceuticals.

A sensitive mutation screening strategy for Fabry disease: detection of nine mutations in the alpha-galactosidase A gene.

Fabry disease is an X-linked recessive lysosomal storage disorder caused by a deficiency of alpha-galactosidase A (alpha-gal; EC 3.2.1.22). In the past, it has been difficult to give an unequivocal diagnosis of carrier status in Fabry disease because of the overlap between normal and heterozygote enzyme levels. To facilitate rapid and accurate carrier and hemizygote detection, a mutation detection strategy was devised to determine the lesion in our Fabry disease patients. The seven alpha-gal exons and adjacent intron boundaries from a representative member of each kindred were PCR amplified and analysed for the presence of sequence alterations by single-stranded conformation polymorphism (SSCP) analysis followed by PCR sequencing. Here we report the use of this strategy in the detection and analysis of the causative mutations in 9 patients with classic severe Fabry disease. Three deletions of 1-, 2-, and 3-bp (987delC, 717delAA, and delta E358), five amino acid substitutions (C52R, G128E, P205T, M284T, and N298K) and a mutation that affects the initiating methionine (M1I) were found in these patients. Counting a previously reported mutation, this strategy has now successfully detected all the Fabry disease mutations present in the 10 kindreds that have been analysed.

Cloning of the sulphamidase gene and identification of mutations in Sanfilippo A syndrome.

Sanfilippo A syndrome is one of four recognised Sanfilippo sub-types (A, B, C and D) that result from deficiencies of different enzymes involved in the lysosomal degradation of heparan sulphate; patients suffer from severe neurological disorders. The Sanfilippo syndrome sub-types are also known as mucopolysaccharidosis (MPS) type III (MPS-IIIA, B, C and D), and are part of the large group of lysosomal storage disorders. Each of the MPS-III types is inherited as an autosomal recessive disorder with considerable variation in severity of clinical phenotype. The incidence of Sanfilippo syndrome has been estimated at 1:24,000 in The Netherlands with MPS IIIA (MIM #252900) the most common. MPS-IIIA is the predominant MPS-III in the United Kingdom, and has a similar high incidence to that found in The Netherlands (E. Wraith, personal communication). There is a particularly high incidence of a clinically severe form of MPS-IIIA in the Cayman Islands with a carrier frequency of 0.1 (ref. 4). Due to the mild somatic disease compared to other MPS disorders there is difficulty in diagnosing mild cases of MPS-III, hence Sanfilippo syndrome may be underdiagnosed, especially in patients with mild mental retardation. Here, we report the isolation, sequence and expression of cDNA clones encoding the enzyme sulphamidase (EC 3.10.1.1). In addition, we report the chromosomal localisation of the sulphamidase gene as being 17q25.3. An 11-bp deletion, present in sulphamidase cDNA from two unrelated Sanfilippo A patients, is described.

Expression, purification and characterization of recombinant human N-acetylgalactosamine-6-sulphatase.

Full-length cDNA sequences encoding human N-acetylgalactosamine-6-sulphatase were stably expressed in Chinese hamster ovary cells under the transcriptional control of the human polypeptide chain elongation factor 1 alpha gene promoter. A clonal cell line overexpressing recombinant N-acetylgalactosamine-6-sulphatase to a level of approx. 3 mg/l of culture medium was isolated. The secreted precursor enzyme was purified to homogeneity by a two-column procedure with an overall yield of 53% of the activity. The physical and catalytic parameters of the recombinant enzyme were similar to those of the mature form isolated from liver. On SDS/PAGE and gel filtration, recombinant N-acetylgalactosamine-6-sulphatase had a native molecular mass of 58-60 kDa. Recombinant N-acetylgalactosamine-6-sulphatase was endocytosed by mucopolysaccharidosis IVA fibroblasts via the mannose-6-phosphate receptor-mediated pathway and was efficiently localized to lysosomes.