Alasdair MacKenzie

Genome-Wide Association Study of Major Recurrent Depression in the U.K. Population

OBJECTIVEnStudies of major depression in twins and families have shown moderate to high heritability, but extensive molecular studies have failed to identify susceptibility genes convincingly. To detect genetic variants contributing to major depression, the authors performed a genome-wide association study using 1,636 cases of depression ascertained in the U.K. and 1,594 comparison subjects screened negative for psychiatric disorders.nnnMETHODnCases were collected from 1) a case-control study of recurrent depression (the Depression Case Control [DeCC] study; N=1346), 2) an affected sibling pair linkage study of recurrent depression (probands from the Depression Network [DeNT] study; N=332), and 3) a pharmacogenetic study (the Genome-Based Therapeutic Drugs for Depression [GENDEP] study; N=88). Depression cases and comparison subjects were genotyped at Centre National de Génotypage on the Illumina Human610-Quad BeadChip. After applying stringent quality control criteria for missing genotypes, departure from Hardy-Weinberg equilibrium, and low minor allele frequency, the authors tested for association to depression using logistic regression, correcting for population ancestry.nnnRESULTSnSingle nucleotide polymorphisms (SNPs) in BICC1 achieved suggestive evidence for association, which strengthened after imputation of ungenotyped markers, and in analysis of female depression cases. A meta-analysis of U.K. data with previously published results from studies in Munich and Lausanne showed some evidence for association near neuroligin 1 (NLGN1) on chromosome 3, but did not support findings at BICC1.nnnCONCLUSIONSnThis study identifies several signals for association worthy of further investigation but, as in previous genome-wide studies, suggests that individual gene contributions to depression are likely to have only minor effects, and very large pooled analyses will be required to identify them.

Differential Activity by Polymorphic Variants of a Remote Enhancer that Supports Galanin Expression in the Hypothalamus and Amygdala: Implications for Obesity, Depression and Alcoholism

The expression of the galanin gene (GAL) in the paraventricular nucleus (PVN) and in the amygdala of higher vertebrates suggests the requirement for highly conserved, but unidentified, regulatory sequences that are critical to allow the galanin gene to control alcohol and fat intake and modulate mood. We used comparative genomics to identify a highly conserved sequence that lay 42u2009kb 5′ of the human GAL transcriptional start site that we called GAL5.1. GAL5.1 activated promoter activity in neurones of the PVN, arcuate nucleus and amygdala that also expressed the galanin peptide. Analysis in neuroblastoma cells demonstrated that GAL5.1 acted as an enhancer of promoter activity after PKC activation. GAL5.1 contained two polymorphisms; rs2513280(C/G) and rs2513281(A/G), that occurred in two allelic combinations (GG or CA) where the dominant GG alelle occurred in 70-83u2009% of the human population. Intriguingly, both SNPs were found to be in LD (R2 of 0.687) with another SNP (rs2156464) previously associated with major depressive disorder (MDD). Recreation of these alleles in reporter constructs and subsequent magnetofection into primary rat hypothalamic neurones showed that the CA allele was 40u2009% less active than the GG allele. This is consistent with the hypothesis that the weaker allele may affect food and alcohol preference. The linkage of the SNPs analysed in this study with a SNP previously associated with MDD together with the functioning of GAL5.1 as a PVN and amygdala specific enhancer represent a significant advance in our ability to understand alcoholism, obesity and major depressive disorder.

The human preprotachykinin-A gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS

Toward an understanding of the mechanisms controlling Preprotachykinin-A (PPTA) transcription, we introduced a 380-kb human yeast artificial chromosome containing the PPTA gene tagged with the beta-galactosidase gene into transgenic mice. This resulted in a pattern of LacZ expression in the central nervous system (CNS) remarkably similar to that reported for PPTA mRNA in the rat. However, the human gene drove expression in areas of the mouse CNS not associated with strong PPTA expression in rodents but which have been shown to express PPTA in the human. This study clearly demonstrates the high degree of conservation of the mechanisms involved in PPTA transcription that has occurred throughout 100 million of divergent human and rodent evolution. This study also defines the maximum linear extent of the human PPT-A promoter. We believe these findings constitute the removal of a significant obstacle in studying the transcriptional regulation of the human PPTA gene in vivo.

Molecular models to analyse preprotachykinin-A expression and function

Towards an understanding of the mechanisms controlling Preprotachykinin A (PPT) expression we have generated a variety of molecular models to determine the mechanisms regulating both the tissue-specific and stimulus-inducible expression of the PPT gene. The approaches used include transgenic and virus vector models complementing biochemical analysis of promoter interactions with transcription factors. We have identified and characterised a yeast artificial chromosome (YAC) containing the human PPT gene and generated transgenic mouse lines containing multiple copies of this chromosome on a normal mouse genetic background. This resulted in a pattern of expression in the nervous system remarkably similar to that reported for PPT mRNA in rodents. In addition, this transgenic model has been constructed in such a manner to allow for over expression of tachykinins based on the number of extra alleles in the transgenic mouse. These animals allow us to further examine the function of the tachykinins and acts as a useful complement to existing PPT ablated mice. In vitro we have introduced the proximal PPT promoter in reporter gene constructs into adult neurones in both DRG and the CNS by an adenoassociated virus (AAV) vector or by biolistic transfection respectively. Using the AAV vector we have demonstrated that the proximal promoter can mediate the effects of NGF in adult rat DRG. These models allow us to delineate transcriptional domains involved in the physiological and pathological expression of the PPT gene.

Allele-specific differences in activity of a novel cannabinoid receptor 1 (CNR1) gene intronic enhancer in hypothalamus, dorsal root ganglia, and hippocampus.

Background: Intron 2 of CNR1 gene contains multiple disease-associated SNPs. Results: Allelic variants of a novel enhancer in CNR1 intron 2 affect its MAPK response in hypothalamus and hippocampus. Conclusion: Alleles of enhancer may be functionally linked to obesity and addictive behavior. Significance: Understanding the effects of CNR1 polymorphisms on gene regulation will accelerate understanding of human disease. Polymorphisms within intron 2 of the CNR1 gene, which encodes cannabinoid receptor 1 (CB1), have been associated with addiction, obesity, and brain volume deficits. We used comparative genomics to identify a polymorphic (rs9444584-C/T) sequence (ECR1) in intron 2 of the CNR1 gene that had been conserved for 310 million years. The C-allele of ECR1 (ECR1(C)) acted as an enhancer in hypothalamic and dorsal root ganglia cells and responded to MAPK activation through the MEKK pathway but not in hippocampal cells. However, ECR1(T) was significantly more active in hypothalamic and dorsal root ganglia cells but, significantly, and in contrast to ECR1(C), was highly active in hippocampal cells where it also responded strongly to activation of MAPK. Intriguingly, rs9444584 is in strong linkage disequilibrium with two other SNPs (rs9450898 (r2 = 0.841) and rs2023239 (r2 = 0.920)) that have been associated with addiction, obesity (rs2023239), and reduced fronto-temporal white matter volumes in schizophrenia patients as a result of cannabis misuse (rs9450898). Considering their high linkage disequilibrium and the increased response of ECR1(T) to MAPK signaling when compared with ECR1(C), it is possible that the functional effects of the different alleles of rs9444584 may play a role in the conditions associated with rs9450898 and rs2023239. Further analysis of the different alleles of ECR1 may lead to a greater understanding of the role of CNR1 gene misregulation in these conditions as well as chronic inflammatory pain.

Understanding the Dynamics of Gene Regulatory Systems; Characterisation and Clinical Relevance of cis-Regulatory Polymorphisms

Modern genetic analysis has shown that most polymorphisms associated with human disease are non-coding. Much of the functional information contained in the non-coding genome consists of cis-regulatory sequences (CRSs) that are required to respond to signal transduction cues that direct cell specific gene expression. It has been hypothesised that many diseases may be due to polymorphisms within CRSs that alter their responses to signal transduction cues. However, identification of CRSs, and the effects of allelic variation on their ability to respond to signal transduction cues, is still at an early stage. In the current review we describe the use of comparative genomics and experimental techniques that allow for the identification of CRSs building on recent advances by the ENCODE consortium. In addition we describe techniques that allow for the analysis of the effects of allelic variation and epigenetic modification on CRS responses to signal transduction cues. Using specific examples we show that the interactions driving these elements are highly complex and the effects of disease associated polymorphisms often subtle. It is clear that gaining an understanding of the functions of CRSs, and how they are affected by SNPs and epigenetic modification, is essential to understanding the genetic basis of human disease and stratification whilst providing novel directions for the development of personalised medicine.

Solvent and additive interactions as determinants in the nucleation pathway: general discussion

Sarah Price opened a general discussion of the paper by Sven Schroeder: I have been generating the thermodynamically plausible crystal structures of organic molecules for many years, and back in 2004 we did a crystal structure prediction (CSP) study on imidazole1 and found that it was relatively straightforward. Following your paper, we have reclassified the low energy structures according to the tilt within the hydrogen-bonded chain and the relative direction of the chains. Although the observed structure was the global minimum, two other structures with a displacement of otherwise identical layers are very close in energy. Do you think that if imidazole had crystallised in one of these alternative structures it would be distinguishable by NEXAFS? This would be a very sensitive test of whether NEXAFS combined with CSP could be used in characterising crystal structures.