Laura M. Fiori

MEIS1 intronic risk haplotype associated with restless legs syndrome affects its mRNA and protein expression levels

Restless legs syndrome (RLS) is a common neurological disorder characterized by an irresistible urge to move the legs at night, which is often accompanied by unpleasant sensations. A recent genomewide association study identified an association between RLS and intronic markers from the MEIS1 gene. Comparative genomic analysis indicates that MEIS1 is the only gene encompassed in this evolutionarily conserved chromosomal segment, i.e. a conservation synteny block, from mammals to fish. We carried out a series of experiments to delineate the role of MEIS1 in RLS pathogenesis and the underlying genetic mechanism. We sequenced all 13 MEIS1 exons and their splice junctions in 285 RLS probands with confirmed clinical diagnosis and did not identify any causative coding or exon-intron junction mutations. We found no evidence of structural variation or disease-associated haplotype differential splicing. However, sequencing of conserved regions of MEIS1 introns 8 and 9 identified a novel single nucleotide polymorphism (C13B_2) significantly associated with RLS (allelic association, P = 1.81E-07). We detected a significant decrease in MEIS1 mRNA expression by quantitative real-time polymerase chain reaction in lymphoblastoid cell lines (LCLs) and brain tissues from RLS patients homozygous for the intronic RLS risk haplotype, compared with those homozygous for the non-risk haplotype. Finally, we found significantly decreased MEIS1 protein levels in the same batch of LCLs and brain tissues from the homozygous carriers of the risk haplotype, compared with the homozygous non-carriers. Therefore, these data suggest that reduced expression of the MEIS1 gene, possibly through intronic cis-regulatory element(s), predisposes to RLS.

Profiling brain expression of the spermidine/spermine N1-acetyltransferase 1 (SAT1) gene in suicide.

Altered stress reactivity is considered to be a risk factor for both major depressive disorder and suicidal behavior. The authors have sought to expand their previous findings implicating altered expression of spermidine/spermine N1‐acetyltransferase 1 (SAT1), the rate‐limiting enzyme involved in catabolism of the polyamines spermidine and spermine in the polyamine stress response (PSR), across multiple brain regions between control individuals and depressed individuals who have died by suicide. Microarray expression of probesets annotated to SAT1 were examined across 17 brain regions in 13 controls and 26 individuals who have died by suicide (16 with a diagnosis of major depression and 10 without), all of French‐Canadian origin. Profiling conducted on the Affymetrix U133A/B chipset was further examined on a second chipset (U133 Plus 2.0) using RT‐PCR, and analyzed in a second, independent sample. A reduction in SAT1 expression identified through multiple probesets was observed across 12 cortical regions in depressed individuals who have died by suicide compared with controls. Of these, five cortical regions showed statistically significant reductions which were supported by RT‐PCR and analysis on the additional chipset. SAT1 cortical expression levels were also found to be significantly lower in an independent sample of German subjects with major depression who died by suicide in comparison with controls. These findings suggest that downregulation of SAT1 expression may play a role in depression and suicidality, possibly by impeding the normal PSR program or through compensation for the increased polyamine metabolism accompanying the psychological distress associated with depressive disorders.

Biomarker discovery: quantification of microRNAs and other small non-coding RNAs using next generation sequencing

BackgroundSmall ncRNAs (sncRNAs) offer great hope as biomarkers of disease and response to treatment. This has been highlighted in the context of several medical conditions such as cancer, liver disease, cardiovascular disease, and central nervous system disorders, among many others. Here we assessed several steps involved in the development of an ncRNA biomarker discovery pipeline, ranging from sample preparation to bioinformatic processing of small RNA sequencing data.MethodsA total of 45 biological samples were included in the present study. All libraries were prepared using the Illumina TruSeq Small RNA protocol and sequenced using the HiSeq2500 or MiSeq Illumina sequencers. Small RNA sequencing data was validated using qRT-PCR. At each stage, we evaluated the pros and cons of different techniques that may be suitable for different experimental designs. Evaluation methods included quality of data output in relation to hands-on laboratory time, cost, and efficiency of processing.ResultsOur results show that good quality sequencing libraries can be prepared from small amounts of total RNA and that varying degradation levels in the samples do not have a significant effect on the overall quantification of sncRNAs via NGS. In addition, we describe the strengths and limitations of three commercially available library preparation methods: (1) Novex TBE PAGE gel; (2) Pippin Prep automated gel system; and (3) AMPure XP beads. We describe our bioinformatics pipeline, provide recommendations for sequencing coverage, and describe in detail the expression and distribution of all sncRNAs in four human tissues: whole-blood, brain, heart and liver.ConclusionsUltimately this study provides tools and outcome metrics that will aid researchers and clinicians in choosing an appropriate and effective high-throughput sequencing quantification method for various study designs, and overall generating valuable information that can contribute to our understanding of small ncRNAs as potential biomarkers and mediators of biological functions and disease.

Genetic and epigenetic influences on expression of spermine synthase and spermine oxidase in suicide completers

Alterations in the levels of spermine synthase (SMS) and spermine oxidase (SMOX), two enzymes involved in polyamine metabolism, have previously been observed in brains of suicide completers. To characterize the roles played by genetic and epigenetic factors in determining expression levels of these genes, as well as to identify potential mechanisms by which to explain our findings in suicide completers, we (1) assessed the role of promoter polymorphisms in determining expression in the brain and in vitro, and (2) examined CpG methylation and levels of methylated histone H3 lysine-27 in the promoter regions of these genes in the prefrontal cortex of suicide completers and healthy controls. We identified several promoter haplotypes in SMS and SMOX, but found no consistent effects of haplotype on expression levels in either the brain or in reporter gene assays performed in three different cell lines. We also found no overall effects of epigenetic factors in determining expression, with the exception of a relationship between CpG methylation at one site in the promoter of SMOX and its expression in Brodmann area 8/9. In conclusion, the genetic and epigenetic factors examined in this study show little influence on the expression levels of SMS and SMOX, and do not appear to be responsible for the dysregulated expression of these genes in suicide completers.

Global gene expression profiling of the polyamine system in suicide completers

In recent years, gene expression, genetic association, and metabolic studies have implicated the polyamine system in psychiatric conditions, including suicide. Given the extensive regulation of genes involved in polyamine metabolism, as well as their interconnections with the metabolism of other amino acids, we were interested in further investigating the expression of polyamine-related genes across the brain in order to obtain a more comprehensive view of the dysregulation of this system in suicide. To this end, we examined the expression of genes related to polyamine metabolism across 22 brain regions in a sample of 29 mood-disordered suicide completers and 16 controls, and identified 14 genes displaying differential expression. Among these, altered expression of spermidine/spermine N1-acetyltransferase, spermine oxidase, and spermine synthase, has previously been observed in brains of suicide completers, while the remainder of the genes represent novel findings. In addition to genes with direct involvement in polyamine metabolism, including S-adenosylmethionine decarboxylase, ornithine decarboxylase antizymes 1 and 2, and arginase II, we identified altered expression of several more distally related genes, including aldehyde dehydrogenase 3 family, member A2, brain creatine kinase, mitochondrial creatine kinase 1, glycine amidinotransferase, glutamic-oxaloacetic transaminase 1, and arginyl-tRNA synthetase-like. Many of these genes displayed altered expression across several brain regions, strongly implying that dysregulated polyamine metabolism is a widespread phenomenon in the brains of suicide completers. This study provides a broader view of the nature and extent of the dysregulation of the polyamine system in suicide, and highlights the importance of this system in the neurobiology of suicide.

Effects of histone modifications on increased expression of polyamine biosynthetic genes in suicide

Altered polyamine metabolism has been consistently observed as underlying the suicide process. We recently performed a global analysis of polyamine gene expression across the brains of suicide completers, and identified up-regulation of four genes, arginase II (ARG2), S-adenosylmethionine decarboxylase (AMD1), and antizymes 1 and 2 (OAZ1 and OAZ2), which play essential roles in polyamine biosynthesis. To determine if a shared epigenetic mechanism is involved in their overexpression in the prefrontal cortex, we measured promoter levels of tri-methyl modified histone-3-lysine-4 (H3K4me3), a marker of open chromatin, and assessed its association with suicide and gene expression. We identified increased H3K4me3 in the promoter region of OAZ1 in suicide, and found that H3K4me3 was correlated with the expression of OAZ1 and ARG2. Overall, our findings indicate that the H3K4me3 modification plays an important role in the regulation of polyamine biosynthesis, and that this mechanism may be involved in the neurobiology of suicide.

Evidence of Altered Polyamine Concentrations in Cerebral Cortex of Suicide Completers

Recent studies have implicated alterations in the expression of polyamine-related genes in the brains of suicide completers including widespread downregulation of spermidine/spermine N1-acetyltransferase, the key enzyme in polyamine catabolism, suggesting compensatory mechanisms attempting to increase brain levels of polyamines. Given the complexity of the polyamine system, quantification of the levels of the polyamines is an essential step in understanding the downstream effects of dysregulated gene expression. We developed a method using high-resolution capillary gas chromatography (GC) in combination with mass spectrometry (MS) for quantitation of polyamines from post-mortem brain tissue, which allowed us to accurately measure spermidine and putrescine concentrations in post-mortem brain tissues. Using this method, we analyzed putrescine and spermidine levels in a total of 126 samples from Brodmann areas 4, 8/9, and 11, from 42 subjects, comprising 16 suicide completers with major depression, 13 non-depressed suicide completers, and 13 control subjects. Both putrescine and spermidine levels fell within the expected nanomolar ranges and were significantly elevated in the brain of suicide completers with a history of major depression as compared with controls. These results were not accounted by possible confounders. This is the first GC–MS study to analyze the expression of putrescine and spermidine from post-mortem brain tissue and confirms the hypothesis raised by previous studies indicating alterations in putrescine and spermidine levels in suicide/major depression.

Epigenetic regulation of spermidine/spermine N1-acetyltransferase (SAT1) in Suicide

We have recently shown that the expression of spermidine/spermine N1-acetyltransferase (SAT1) is downregulated across the brains of suicide completers, and that its expression is influenced by genetic variations in the promoter. Several promoter polymorphisms in SAT1, including rs6526342, have been associated with suicide and other psychiatric disorders, and display haplotype-specific effects on expression. However, these effects cannot explain total variability in SAT1 expression, and other regulatory mechanisms, such as epigenetic factors, may also be at play. In this study, we assessed the involvement of epigenetic factors in controlling SAT1 expression in the prefrontal cortex of suicide completers by mapping CpG methylation across a 1880-bp region of the SAT1 promoter, and measuring levels of tri-methylated histone-3-lysine 27 (H3K27me3) at the promoter in suicide completers and controls. Our results demonstrated that CpG methylation was significantly negatively correlated with SAT1 expression. Although overall or site-specific CpG methylation was not associated with suicide or SAT1 expression, we observed high levels of methylation at the polymorphic CpG site created by rs6526342, indicating a relationship between promoter haplotypes and methylation. There was no association between H3K27me3 and suicide, nor was this modification associated with SAT1 expression. Overall, our results indicate that epigenetic factors in the promoter region of SAT1 influence gene expression levels, and may provide a mechanism for both our previous findings of haplotype-specific effects of promoter variations on SAT1 expression, as well as the widespread downregulation of SAT1 expression observed in the brains of suicide completers.

Identification and characterization of spermidine/spermine N1-acetyltransferase promoter variants in suicide completers.

BACKGROUND We have previously shown that the expression of spermidine/spermine N1-acetyltransferase (SAT1) is decreased in the brain Brodmann areas (BA)4, BA8/9, and BA11 of suicide completers and found an association between rs6526342, a SAT1 promoter single nucleotide polymorphism (SNP), with suicide completion (1). METHODS We genotyped 18 promoter polymorphisms in SAT1 in a French-Canadian population. The relationship between haplotypes and gene expression was assessed with microarray analysis of three brain regions as well as reporter gene assays in three cell lines. Site-directed mutagenesis was used to examine the role of individual polymorphisms in reporter gene expression. RESULTS We identified two major and several minor haplotypes in the promoter region of SAT1. Subjects who possessed the haplotype containing the risk allele for rs6526342 demonstrated decreased SAT1 expression in BA4, BA8/9, and BA11. This haplotype was also associated with decreased expression in reporter gene assays. Site-directed mutagenesis identified three polymorphisms-an insertion/deletion (rs6151267), and two SNPs (rs6526342 and rs928931)-that were involved in determining reporter gene expression. These polymorphisms do not seem to exert their effects through the polyamine responsive element, because all constructs were induced to a similar extent in the presence of spermine. CONCLUSIONS Our results indicate that genetic variations in the promoter region of SAT1 are involved in determining levels of gene expression and might provide a mechanism for the decreased SAT1 expression observed in suicide completers.

Association of Polyaminergic Loci With Anxiety, Mood Disorders, and Attempted Suicide

Background The polyamine system has been implicated in a number of psychiatric conditions, which display both alterations in polyamine levels and altered expression of genes related to polyamine metabolism. Studies have identified associations between genetic variants in spermidine/spermine N1-acetyltransferase (SAT1) and both anxiety and suicide, and several polymorphisms appear to play important roles in determining gene expression. Methodology/Principal Findings We genotyped 63 polymorphisms, spread across four polyaminergic genes (SAT1, spermine synthase (SMS), spermine oxidase (SMOX), and ornithine aminotransferase like-1 (OATL1)), in 1255 French-Canadian individuals who have been followed longitudinally for 22 years. We assessed univariate associations with anxiety, mood disorders, and attempted suicide, as assessed during early adulthood. We also investigated the involvement of gene-environment interactions in terms of childhood abuse, and assessed internalizing and externalizing symptoms as endophenotypes mediating these interactions. Overall, each gene was associated with at least one main outcome: anxiety (SAT1, SMS), mood disorders (SAT1, SMOX), and suicide attempts (SAT1, OATL1). Several SAT1 polymorphisms displayed disease-specific risk alleles, and polymorphisms in this gene were involved in gene-gene interactions with SMS to confer risk for anxiety disorders, as well as gene-environment interactions between childhood physical abuse and mood disorders. Externalizing behaviors demonstrated significant mediation with regards to the association between OATL1 and attempted suicide, however there was no evidence that externalizing or internalizing behaviors were appropriate endophenotypes to explain the associations with mood or anxiety disorders. Finally, childhood sexual abuse did not demonstrate mediating influences on any of our outcomes. Conclusions/Significance These results demonstrate that genetic variants in polyaminergic genes are associated with psychiatric conditions, each of which involves a set of separate and distinct risk alleles. As several of these polymorphisms are associated with gene expression, these findings may provide mechanisms to explain the alterations in polyamine metabolism which have been observed in psychiatric disorders.