An exploratory metabolomic comparison of participants with fast or absent functional progression from 2CARE, a randomized, double-blind clinical trial in Huntington's disease.

Huntington's disease (HD) is increasingly recognized for diverse pathology outside of the nervous system. To describe the biology of HD in relation to functional progression, we previously analyzed the plasma and CSF metabolome in a cross-sectional study of participants who had various degrees of functional impairment. Here, we carried out an exploratory study in plasma from HD individuals over a 3-year time frame to assess whether differences exist between those with fast or absent clinical progression. There were more differences in circulating metabolite levels for fast progressors compared to absent progressors (111 vs 20, nominal p < 0.05). All metabolite changes in faster progressors were decreases, whereas some metabolite concentrations increased in absent progressors. Many of the metabolite levels that decreased in the fast progressors were higher at Screening compared to absent progressors but ended up lower by Year 3. Changes in faster progression suggest greater oxidative stress and inflammation (kynurenine, diacylglycerides, cysteine), disturbances in nitric oxide and urea metabolism (arginine, citrulline, ornithine, GABR), lower polyamines (putrescine and spermine), elevated glucose, and deficient AMPK signaling. Metabolomic differences between fast and absent progressors suggest the possibility of predicting functional decline in HD, and possibly delaying it with interventions to augment arginine, polyamines, and glucose regulation.

Reading LINEs within the cocaine addicted brain.

INTRODUCTION: Long interspersed element (LINE)-1 (L1) is a type of retrotransposon capable of mobilizing into new genomic locations. Often studied in Mendelian diseases or cancer, L1s may also cause somatic mutation in the developing central nervous system. Recent reports showed L1 transcription was activated in brains of cocaine-treated mice, and L1 retrotransposition was increased in cocaine-treated neuronal cell cultures. We hypothesized that the predisposition to cocaine addiction may result from inherited L1s or somatic L1 mobilization in the brain. METHODS: Postmortem medial prefrontal cortex (mPFC) tissue from 30 CA and 30 control individuals was studied. An Alexafluor488-labeled NeuN antibody and fluorescence activated nuclei sorting were used to separate neuronal from non-neuronal cell nuclei. L1s and their 3' flanking sequences were amplified from neuronal and non-neuronal genomic DNA (gDNA) using L1-seq. L1 DNA libraries from the neuronal gDNA were sequenced on an Illumina HiSeq2000. Sequences aligned to the hg19 human genome build were analyzed for L1 insertions using custom "L1-seq" bioinformatics programs. RESULTS: Previously uncataloged L1 insertions, some validated by PCR, were detected in neurons from both CA and control brain samples. Steady-state L1 mRNA levels in CA and control mPFC were also assessed. Gene ontology and pathway analyses were used to assess relationships between genes putatively disrupted by novel L1s in CA and control individuals. L1 insertions in CA samples were enriched in gene ontologies and pathways previously associated with CA. CONCLUSIONS: We conclude that neurons in the mPFC harbor L1 insertions that have the potential to influence predisposition to CA.

Low frequency genetic variants in the µ-opioid receptor (OPRM1) affect risk for addiction to heroin and cocaine.

The µ-opioid receptor (MOR) binds exogenous and endogenous opioids and is known to mediate the rewarding effects of drugs of abuse. Numerous genetic studies have sought to identify common genetic variation in the gene encoding MOR (OPRM1) that affects risk for drug addiction. The purpose of this study was to examine the contribution of rare coding variants in OPRM1 to the risk for addiction. Rare and low frequency variants were selected using the National Heart Lung and Blood Institute - Exome Sequencing Project (NHLBI-ESP) database, which has screened the exomes of over 6500 individuals. Two SNPs (rs62638690 and rs17174794) were selected for genotyping in 1377 European American individuals addicted to heroin and/or cocaine. Two different SNPs (rs1799971 and rs17174801) were genotyped in 1238 African American individuals addicted to heroin and/or cocaine. Using the minor allele frequencies from the NHLBI-ESP dataset as a comparison group, case-control association analyses were performed. Results revealed an association between rs62638690 and cocaine and heroin addiction in European Americans (p=0.02; 95% C.I. 0.47 [0.24-0.92]). This study suggests a potential role for rare OPRM1 variants in addiction disorders and highlights an area worthy of future study.

In vitro and ex vivo analysis of CHRNA3 and CHRNA5 haplotype expression.

Genome-wide association studies implicate variations in CHRNA5 and CHRNA3 as being associated with nicotine addiction (NA). Multiple common haplotypes ("risk", "mixed" and "protective") exist in Europeans; however, high linkage disequilibrium between variations in CHRNA5 and CHRNA3 makes assigning causative allele(s) for NA difficult through genotyping experiments alone. We investigated whether CHRNA5 or CHRNA3 promoter haplotypes, associated previously with NA, might influence allelic expression levels. For in vitro analyses, promoter haplotypes were sub-cloned into a luciferase reporter vector. When assessed in BE(2)-C cells, luciferase expression was equivalent among CHRNA3 haplotypes, but the combination of deletion at rs3841324 and variation at rs503464 decreased CHRNA5 promoter-derived luciferase activity, possibly due to loss of an SP-1 and other site(s). Variation within the CHRNA5 5'UTR at rs55853698 and rs55781567 also altered luciferase expression in BE(2)-C cells. Allelic expression imbalance (AEI) from the "risk" or "protective" haplotypes was assessed in post-mortem brain tissue from individuals heterozygous at coding polymorphisms in CHRNA3 (rs1051730) or CHRNA5 (rs16969968). In most cases, equivalent allelic expression was observed; however, one individual showed CHRNA5 AEI that favored the "protective" allele and that was concordant with heterozygosity at polymorphisms ∼13.5 kb upstream of the CHRNA5 transcription start site. Putative enhancer activity from these distal promoter elements was assessed using heterologous promoter constructs. We observed no differences in promoter activity from the two distal promoter haplotypes examined, but found that the distal promoter region strongly repressed transcription. We conclude that CHRNA5 promoter variants may affect relative risk for NA in some heterozygous individuals.

Association analysis of the pituitary adenylate cyclase-activating polypeptide (PACAP/ADCYAP1) gene in bipolar disorder.

BACKGROUND: Linkage studies in bipolar disorder (BPD) suggest that a susceptibility locus exists on chromosome 18p11. The pituitary adenylate cyclase-activating polypeptide/adenylate cyclase-activating polypeptide 1 (pituitary) (PACAP/ADCYAP1) gene maps to this region. PACAP is a neuropeptide involved in neurotransmission in both the peripheral nervous system and central nervous system and is required for catecholamine secretion. Animal models of PACAP mutations show remarkable behavioral defects, including hyperactivity and increased exploratory behavior. OBJECTIVE: In this study we tested the hypothesis that genetic variations in the human PACAP gene contribute to BPD. METHODS: Genotyping of seven single nucleotide polymorphisms (rs1893154; rs2846811; rs8192595; rs2856966; rs928978; rs2231187; rs1610037) was performed in BPD patients (n=570) and healthy controls (n=710). Genotypes and allele frequencies were compared between groups using chi contingency analysis. Linkage disequilibrium between markers was calculated and estimated haplotype frequencies were compared between groups. MAIN RESULTS: There were no significant differences between groups on the allele, genotype or haplotype level for any of the tested single nucleotide polymorphisms. CONCLUSION: Our results provide no evidence of an association of the PACAP gene with BPD in this group of patients and controls. Additional studies are necessary to elucidate the BPD susceptibility locus on chromosome 18p.

Novel de novo mutation of a conserved SCN1A amino-acid residue (R1596).

We report on the case of a 6-year-old boy with epilepsy involving febrile seizures and unprovoked generalized tonic clonic seizures. Genetic testing revealed a novel de novo mutation in the SCN1A gene (C>T 4786, R1596C). The epilepsy phenotype is within the spectrum of generalized epilepsy with febrile seizures plus. However, de novo mutations are more commonly reported in cases of severe myoclonic epilepsy of infancy, and are less often reported in generalized epilepsy with febrile seizures plus. The clinical utility of specific genetic testing in this case is discussed, as are criteria for determining the pathologic significance of novel DNA variants. In this case, the wild type of residue (R1596) is well-conserved across evolution from bacteria to humans, providing support for the hypothesis that this mutation causes epilepsy.

Identification and functional significance of polymorphisms in the mu-opioid receptor gene (Oprm) promoter of C57BL/6 and DBA/2 mice.

C57BL/6J and DBA/2J mice demonstrate differences in morphine preference when tested in a two-bottle choice paradigm. Quantitative trait loci (QTL) mapping suggested the proximal region of chromosome 10 was responsible for 41% of the observed genetic variance. The mu-opioid receptor (MOR) gene (Oprm) maps to this region and is a prime candidate for explaining the QTL. We hypothesized that variations in Oprm between these strains are responsible for differences in morphine preference. We identify five single nucleotide polymorphisms (SNPs) in the Oprm promoter; three within or near putative transcription factor binding sites. Promoter fragments were amplified from genomic DNA by polymerase chain reaction (PCR) and subcloned into luciferase reporter vectors. A significant difference in basal Oprm promoter activity was seen with C57BL/6 and DBA/2 approximately 1675 constructs in MOR-positive BE(2)-C cells, but not in MOR-negative Neuro-2a cells. In BE(2)-C cells, average DBA/2 approximately 1675 construct activity was 1.3-2.0x greater than average C57BL/6 activity suggesting that the SNPs might alter MOR expression in these two mouse strains. Significant differences in promoter activities between the two cell lines suggest that cell-type-specific transcription factors are involved. No significant differences in construct activity were found between untreated and morphine-treated BE(2)-C or Neuro-2a cells, suggesting that morphine does not regulate transcription of Oprm.

No association between common variations in the neuronal nicotinic acetylcholine receptor alpha2 subunit gene (CHRNA2) and bipolar I disorder.

The neuronal nicotinic acetylcholine receptor alpha2 subunit gene (CHRNA2) maps to the bipolar susceptibility locus on chromosome 8p21-22. Given the biological role of the neuronal nicotinic acetylcholine receptors and the substantial comorbidity of nicotine dependence in psychiatric disorders, the CHRNA2 gene is a plausible candidate gene for bipolar disorder (BPD). We tested the hypothesis that variations in the CHRNA2 gene confer susceptibility to bipolar I disorder in a case-control association study. Genotypes of one amino acid substitution polymorphism (Ala125Thr) and five non-coding variations across the CHRNA2 gene were obtained from 345 unrelated bipolar I patients and 273 control samples. Genotypes and allele frequencies were compared between groups using chi-square contingency analysis. Linkage disequilibrium (LD) between markers was calculated, and estimated haplotype frequencies were compared between groups. We observed no statitistically significant difference in genotype and allele frequencies for all six variations between bipolar patients and controls, but we did demonstrate strong LD throughout the gene. Haplotype analysis showed that no combinations of alleles were associated with illness. Our results suggest that common variations in the CHRNA2 gene are unlikely to confer susceptibility to BPD in this sample. Further studies are required to elucidate the susceptibility locus for BPD on chromosome 8p21-22.

No association between common variations in the human alpha 2 subunit gene (ATP1A2) of the sodium-potassium-transporting ATPase and idiopathic generalized epilepsy.

Quantitative trait loci studies in inbred mice have identified a locus on chromosome 1 (Szs1) of fundamental importance to seizure susceptibility. High-ranking candidate genes in this susceptibility region include KCNJ9, KCNJ10 and ATP1A2. We performed a systematic mutation scan of the coding region of the human ATP1A2 gene and performed a case-control association study with seven common markers. Genotypes were assessed in 152 idiopathic generalized epilepsy (IGE) patients of German ancestry and 111 healthy German controls for all seven polymorphisms. No significant differences were found in genotype or allele frequencies for any of the variations between the IGE patients and controls. No haplotypes were associated with IGE when compared to controls. Linkage disequilibrium was demonstrated throughout the gene. Results suggest that the polymorphisms we studied in the ATP1A2 gene do not represent major susceptibility factors for common forms of IGE.