ABSTRACT
Maternal tobacco smoking during pregnancy is a large driver of health inequalities and a higher prevalence of conduct problem (CP) has been observed in exposed offspring. Further, maternal tobacco use during pregnancy can also alter offspring DNA methylation. However, currently, limited molecular evidence has been found to support this observation. Thus we aim to examine the association between maternal tobacco use in pregnancy and offspring CP, to determine whether offspring CP is mediated by tobacco exposure-induced DNA methylation differences. Understanding the etiology of the association between maternal tobacco use and offspring CP will be crucial in the early identification and treatment of CP in children and adolescents. Here, a sub group of N =96 individuals was sourced from the Christchurch Health and Development Study, a longitudinal birth cohort studied for over 40 years in New Zealand. Whole blood samples underwent bisulphite-based amplicon sequencing at 10 loci known to play a role in neurodevelopment, or which had associations with CP phenotypes. We identified significant (P CYP1A1 , ASH2L and MEF2C in individuals with CP who were exposed to tobacco in utero . We conclude that environmentally-induced DNA methylation differences could play a role in the observed link between maternal tobacco use during pregnancy and childhood/adolescent CP. However, larger sample sizes are needed to produce an adequate amount of power to investigate this interaction further.
Subject(s)
DNA Methylation , Prenatal Exposure Delayed Effects , Adolescent , Female , Humans , Pregnancy , Prenatal Exposure Delayed Effects/genetics , Sulfites , Tobacco UseABSTRACT
We present innovative research practices in psychiatric genetic studies to ensure representation of individuals from diverse ancestry, sex assigned at birth, gender identity, age, body shape and size, and socioeconomic backgrounds. Due to histories of inappropriate and harmful practices against marginalized groups in both psychiatry and genetics, people of certain identities may be hesitant to participate in research studies. Yet their participation is essential to ensure diverse representation, as it is incorrect to assume that the same genetic and environmental factors influence the risk for various psychiatric disorders across all demographic groups. We present approaches developed as part of the Eating Disorders Genetics Initiative (EDGI), a study that required tailored approaches to recruit diverse populations across many countries. Considerations include research priorities and design, recruitment and study branding, transparency, and community investment and ownership. Ensuring representation in participants is costly and funders need to provide adequate support to achieve diversity in recruitment in prime awards, not just as supplemental afterthoughts. The need for diverse samples in genetic studies is critical to minimize the risk of perpetuating health disparities in psychiatry and other health research. Although the EDGI strategies were designed specifically to attract and enroll individuals with eating disorders, our approach is broadly applicable across psychiatry and other fields.
Subject(s)
Gender Identity , Research , Female , Humans , Infant, Newborn , MaleABSTRACT
Sodium valproate (VPA) is a histone deacetylase (HDAC) inhibitor, widely prescribed in the treatment of bipolar disorder, and yet the precise modes of therapeutic action for this drug are not fully understood. After exposure of the rat serotonergic cell line RN46A to VPA, RNA-sequencing (RNA-Seq) analysis showed widespread changes in gene expression. Analysis by four bioinformatic pipelines revealed as many as 230 genes were significantly upregulated and 72 genes were significantly downregulated. A subset of 23 differentially expressed genes was selected for validation using the nCounter® platform, and of these we obtained robust validation for ADAM23, LSP1, MAOB, MMP13, PAK3, SERPINB2, SNAP91, WNT6, and ZCCHC12. We investigated the effect of lithium on this subset and found four genes, CDKN1C, LSP1, SERPINB2, and WNT6 co-regulated by lithium and VPA. We also explored the effects of other HDAC inhibitors and the VPA analogue valpromide on the subset of 23 selected genes. Expression of eight of these genes, CDKN1C, MAOB, MMP13, NGFR, SHANK3, VGF, WNT6 and ZCCHC12, was modified by HDAC inhibition, whereas others did not appear to respond to several HDAC inhibitors tested. These results suggest VPA may regulate genes through both HDAC-dependent and independent mechanisms. Understanding the broader gene regulatory effects of VPA in this serotonergic cell model should provide insights into how this drug works and whether other HDAC inhibitor compounds may have similar gene regulatory effects, as well as highlighting molecular processes that may underlie regulation of mood.
Subject(s)
Antimanic Agents/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Histone Deacetylase Inhibitors/pharmacology , Serotonin/metabolism , Transcription, Genetic/genetics , Valproic Acid/pharmacology , Animals , Cell Line , Computational Biology , Histone Deacetylases/metabolism , Lithium/pharmacology , RNA-Seq , Rats , Transcriptional ActivationABSTRACT
BACKGROUND: The Eating Disorders Genetics Initiative (EDGI) is an international investigation exploring the role of genes and environment in anorexia nervosa, bulimia nervosa, and binge-eating disorder. METHODS: A total of 14,500 individuals with eating disorders and 1500 controls will be included from the United States (US), Australia (AU), New Zealand (NZ), and Denmark (DK). In the US, AU, and NZ, participants will complete comprehensive online phenotyping and will submit a saliva sample for genotyping. In DK, individuals with eating disorders will be identified by the National Patient Register, and genotyping will occur using bloodspots archived from birth. A genome-wide association study will be conducted within EDGI and via meta-analysis with other data from the Eating Disorders Working Group of the Psychiatric Genomics Consortium (PGC-ED). DISCUSSION: EDGI represents the largest genetic study of eating disorders ever to be conducted and is designed to rapidly advance the study of the genetics of the three major eating disorders (anorexia nervosa, bulimia nervosa, and binge-eating disorder). We will explicate the genetic architecture of eating disorders relative to each other and to other psychiatric and metabolic disorders and traits. Our goal is for EDGI to deliver "actionable" findings that can be transformed into clinically meaningful insights. TRIAL REGISTRATION: EDGI is a registered clinical trial: clinicaltrials.gov NCT04378101 .
Subject(s)
Bulimia Nervosa , Feeding and Eating Disorders , Australia , Bulimia Nervosa/genetics , Feeding and Eating Disorders/genetics , Genome-Wide Association Study , Humans , Meta-Analysis as Topic , New Zealand , United StatesABSTRACT
BACKGROUND: GBA mutations are numerically the most significant genetic risk factor for Parkinson's disease (PD), yet these mutations have low penetrance, suggesting additional mechanisms. OBJECTIVES: The objective of this study was to determine if the penetrance of GBA in PD can be explained by regulatory effects on GBA and modifier genes. METHODS: Genetic variants associated with the regulation of GBA were identified by screening 128 common single nucleotide polymorphisms (SNPs) in the GBA locus for spatial cis-expression quantitative trail locus (supported by chromatin interactions). RESULTS: We identified common noncoding SNPs within GBA that (1) regulate GBA expression in peripheral tissues, some of which display α-synuclein pathology and (2) coregulate potential modifier genes in the central nervous system and/or peripheral tissues. Haplotypes based on 3 of these SNPs delay disease onset by 5 years. In addition, SNPs on 6 separate chromosomes coregulate GBA expression specifically in either the substantia nigra or cortex, and their combined effect potentially modulates motor and cognitive symptoms, respectively. CONCLUSIONS: This work provides a new perspective on the haplotype-specific effects of GBA and the genetic etiology of PD, expanding the role of GBA from the gene encoding the ß-glucocerebrosidase (GCase) to that of a central regulator and modifier of PD onset, with GBA expression itself subject to distant regulation. Some idiopathic patients might possess insufficient GBA-encoded GCase activity in the substantia nigra as the result of distant regulatory variants and therefore might benefit from GBA-targeting therapeutics. The SNPs' regulatory impacts provide a plausible explanation for the variable phenotypes also observed in GBA-centric Gaucher's disease and dementia with Lewy bodies. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, LLC on behalf of International Parkinson and Movement Disorder Society.
Subject(s)
Gaucher Disease , Parkinson Disease , Gaucher Disease/genetics , Genes, Modifier , Glucosylceramidase/genetics , Humans , Lewy Bodies , Mutation , Parkinson Disease/geneticsABSTRACT
Valproic acid (VPA) and lithium are widely used in the treatment of bipolar disorder. However, the underlying mechanism of action of these drugs is not clearly understood. We used RNA-Seq analysis to examine the global profile of gene expression in a rat serotonergic cell line (RN46A) after exposure to these two mood stabilizer drugs. Numerous genes were differentially regulated in response to VPA (log2 fold change ≥ 1.0; i.e., odds ratio of ≥2, at false discovery rate <5%), but only two genes ( Dynlrb2 and Cdyl2) showed significant differential regulation after exposure of the cells to lithium, with the same analysis criteria. Both of these genes were also regulated by VPA. Many of the differentially expressed genes had functions of potential relevance to mood disorders or their treatment, such as several serpin family genes (including neuroserpin), Nts (neurotensin), Maob (monoamine oxidase B), and Ap2b1, which is important for synaptic vesicle function. Pathway analysis revealed significant enrichment of Gene Ontology terms such as extracellular matrix remodeling, cell adhesion, and chemotaxis. This study in a cell line derived from the raphe nucleus has identified a range of genes and pathways that provide novel insights into potential therapeutic actions of the commonly used mood stabilizer drugs.
Subject(s)
Lithium/pharmacology , Serotonergic Neurons/drug effects , Transcriptome/drug effects , Valproic Acid/pharmacology , Animals , Bipolar Disorder/drug therapy , Cell Adhesion/drug effects , Cell Adhesion/genetics , Cell Line , Chemotaxis/drug effects , Chemotaxis/genetics , Extracellular Matrix/drug effects , Extracellular Matrix/genetics , Gene Ontology , Lithium/therapeutic use , RNA-Seq , Rats , Real-Time Polymerase Chain Reaction , Serotonergic Neurons/metabolism , Transcriptional Activation/drug effects , Transcriptional Activation/genetics , Valproic Acid/therapeutic useABSTRACT
OBJECTIVES: The MinION nanopore sequencing device opens the opportunity to cost-effective and point-of-care DNA sequencing. As a proof of principle, we developed a multiplex assay targeting pharmacogenetic variants related to clopidogrel and warfarin, the two commonly used drugs that show response variability due to genetic polymorphisms. METHODS: Six reference and 78 clinical DNA samples were amplified by PCR to generate 15 amplicons targeting 27 key variants. These products were then barcoded to enable sample multiplexing in one sequencing run. Four variant calling tools (marginCaller, VarScan 2, nanopolish, Clairvoyante) were used to compare genotyping accuracy. RESULTS: In our cohort, 81 out of 84 samples were successfully sequenced and genotyped. Using nanopolish as the variant calling tool achieved accuracy >95% for all except two variants. A known single base deletion (CYP2C9*6) was successfully detected. CONCLUSION: While minor misgenotyping issues exist, this work demonstrates that drug-specific or broad pharmacogenetic screening assays using small PCR amplicons are possible on the MinION sequencing device.
Subject(s)
Nanopore Sequencing/instrumentation , Pharmacogenetics , Genotyping Techniques , Humans , Polymorphism, Single Nucleotide/geneticsABSTRACT
Angioedema is a rare adverse effect of the commonly used angiotensin-converting enzyme inhibitors (ACEi) and is reported to occur with a prevalence of 0.1%-0.7%. Although most ACEi-induced angioedema (ACEi-A) cases are mild, severe cases requiring intensive care and even resulting in death have been reported in the literature. The mechanisms underlying ACEi-A are not yet fully understood, but bradykinin and/or substance P accumulation resulting from inhibition of ACE is believed to play a crucial role. ACEi-A occurs at variable frequencies across different racial groups, suggesting a genetic association with the development of ACEi-A. To date, one genome-wide association study and several candidate gene studies have been published on the association of genetic variation with ACEi-A. Genetic associations reported have been attributed to several distinct mechanisms: (a) genes coding for alternative enzymes responsible for the degradation of bradykinin and/or substance P in the diminution of ACE activity (b) ACE gene function, (c) bradykinin receptor genes, (d) genes implicated in immune and inflammation regulation and (e) genes in the fibrinolytic and coagulation pathway. Despite several plausible genetic associations, there are currently no genetic variants with sufficient effect to be clinically useful. The low incidence of ACEi-A suggests that a combination of genomic approaches with the capability to detect potentially important variants might be required to shed light on the mechanism of this adverse reaction. Additionally, many non-genetic risk factors associated with ACEi-A suggest the potential contribution of epigenetic dysregulation.
Subject(s)
Angioedema , Angiotensin-Converting Enzyme Inhibitors/adverse effects , Polymorphism, Genetic , Angioedema/blood , Angioedema/chemically induced , Angioedema/epidemiology , Angioedema/genetics , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Bradykinin/blood , Bradykinin/genetics , Genome-Wide Association Study , Humans , Pharmacogenetics , Prevalence , Substance P/blood , Substance P/geneticsABSTRACT
Many aspects of human development and disease are influenced by the interaction between genetic and environmental factors. Understanding how our genes respond to the environment is central to managing health and disease, and is one of the major contemporary challenges in human genetics. Various epigenetic processes affect chromosome structure and accessibility of deoxyribonucleic acid (DNA) to the enzymatic machinery that leads to expression of genes. One important epigenetic mechanism that appears to underlie the interaction between environmental factors, including diet, and our genome, is chemical modification of the DNA. The best understood of these modifications is methylation of cytosine residues in DNA. It is now recognized that the pattern of methylated cytosines throughout our genomes (the 'methylome') can change during development and in response to environmental cues, often with profound effects on gene expression. Many dietary constituents may indirectly influence genomic pathways that methylate DNA, and there is evidence for biochemical links between nutritional quality and mental health. Deficiency of both macro- and micronutrients has been associated with increased behavioural problems, and nutritional supplementation has proven efficacious in treatment of certain neuropsychiatric disorders. In this review we examine evidence from the fields of nutrition, developmental biology, and mental health that supports dietary impacts on epigenetic processes, particularly DNA methylation. We then consider whether such processes could underlie the demonstrated efficacy of dietary supplementation in treatment of mental disorders, and whether targeted manipulation of DNA methylation patterns using controlled dietary supplementation may be of wider clinical value.
Subject(s)
Epigenesis, Genetic , Mental Health , Nutritional Status , Animals , DNA Methylation/drug effects , Diet , Dietary Supplements , Disease Models, Animal , Gene Expression Regulation , Humans , Mental Disorders/diagnosis , Mental Disorders/diet therapy , Mental Disorders/genetics , Micronutrients/administration & dosage , Micronutrients/deficiency , Neurodevelopmental Disorders/diagnosis , Neurodevelopmental Disorders/diet therapy , Neurodevelopmental Disorders/geneticsABSTRACT
The promoter of the human imprinted gene MEST is differentially methylated with respect to the parent of origin and contains several non B-DNA motifs that are capable of forming G-quadruplexes. These factors can contribute to a consistent allelic dropout (ADO) of the maternally methylated DNA during polymerase chain reaction (PCR) analysis of such gene regions. Here, we directly investigate the cause of allelic dropout by applying fluorescent techniques to visualize polymerase amplification and arrest during PCR of differentially methylated DNA templates. We demonstrate that polymerase arrest corresponds to previously characterized G-quadruplex-forming motifs at the MEST promoter region and occurs at equivalent sites on both methylated and nonmethylated DNA templates. However, during PCR, polymerase arrest can be observed on the methylated template for several cycles longer than on the nonmethylated template, and this results in an amplification lag and a lower yield of full length amplicons. We demonstrate that this delay in amplification is sufficient to cause complete ADO during PCR, providing a mechanistic basis for the previously observed genotyping error at this locus.
Subject(s)
DNA Methylation , G-Quadruplexes , Polymerase Chain Reaction , Promoter Regions, Genetic , Proteins , HumansABSTRACT
BACKGROUND: Laboratory assays evaluating the effect of DNA sequence variants on BRCA1 mRNA splicing may contribute to classification by providing molecular evidence. However, our knowledge of normal and aberrant BRCA1 splicing events to date has been limited to data derived from assays targeting partial transcript sequences. This study explored the utility of nanopore sequencing to examine whole BRCA1 mRNA transcripts and to provide accurate categorisation of in-frame and out-of-frame splicing events. METHODS: The exon structure of BRCA1 transcripts from a previously studied control lymphoblastoid cell line were assessed using MinION nanopore sequencing of long-range reverse transcriptase-PCR amplicons. RESULTS: Our study identified and characterised 32 complete BRCA1 isoforms, including 18 novel isoforms which showed skipping of multiple contiguous and/or non-contiguous exons. Furthermore, we show that known BRCA1 exon skipping events, such as Δ(9,10) and Δ21, can co-occur in a single transcript, with some isoforms containing four or more alternative splice junctions. Fourteen novel isoforms were formed entirely from a combination of previously identified alternative splice junctions, suggesting that the total number of BRCA1 isoforms might be lower than the number of splicing events reported previously. CONCLUSIONS: Our results highlight complexity in BRCA1 transcript structure that has not been described previously. This finding has key implications for predicting the translation frame of splicing transcripts, important for interpreting the clinical significance of spliceogenic variants. Future research is warranted to quantitatively assess full-length BRCA1 transcript levels, and to assess the application of nanopore sequencing for routine evaluation of potential spliceogenic variants.
Subject(s)
Alternative Splicing , Exons , Genes, BRCA1 , High-Throughput Nucleotide Sequencing , RNA, Messenger/genetics , Base Sequence , Cell Line, Tumor , DNA, Complementary/chemistry , DNA, Complementary/genetics , Female , High-Throughput Nucleotide Sequencing/methods , Humans , Nonsense Mediated mRNA DecayABSTRACT
BACKGROUND: The genetic and environmental influences on human personality and behaviour are a complex matter of ongoing debate. Accumulating evidence indicates that short tandem repeats (STRs) in regulatory regions are good candidates to explain heritability not accessed by genome-wide association studies. METHODS: We tested for associations between the genotypes of four selected repeats and 18 traits relating to personality, behaviour, cognitive ability and mental health in a well-studied longitudinal birth cohort (n = 458-589) using one way analysis of variance. The repeats were a highly conserved poly-AC microsatellite in the upstream promoter region of the T-box brain 1 (TBR1) gene and three previously studied STRs in the activating enhancer-binding protein 2-beta (AP2-ß) and androgen receptor (AR) genes. Where significance was found we used multiple regression to assess the influence of confounding factors. RESULTS: Carriers of the shorter, most common, allele of the AR gene's GGN microsatellite polymorphism had fewer anxiety-related symptoms, which was consistent with previous studies, but in our study this was not significant following Bonferroni correction. No associations with two repeats in the AP2-ß gene withstood this correction. A novel finding was that carriers of the minor allele of the TBR1 AC microsatellite were at higher risk of conduct problems in childhood at age 7-9 (p = 0.0007, which did pass Bonferroni correction). Including maternal smoking during pregnancy (MSDP) in models controlling for potentially confounding influences showed that an interaction between TBR1 genotype and MSDP was a significant predictor of conduct problems in childhood and adolescence (p < 0.001), and of self-reported criminal behaviour up to age 25 years (p ≤ 0.02). This interaction remained significant after controlling for possible confounders including maternal age at birth, socio-economic status and education, and offspring birth weight. CONCLUSIONS: The potential functional importance of the TBR1 gene's promoter microsatellite deserves further investigation. Our results suggest that it participates in a gene-environment interaction with MDSP and antisocial behaviour. However, previous evidence that mothers who smoke during pregnancy carry genes for antisocial behaviour suggests that epistasis may influence the interaction.
Subject(s)
Behavior , Cognition , Microsatellite Repeats/genetics , Adolescent , Adult , Alleles , Child , Criminal Behavior , Female , Gene-Environment Interaction , Genotype , Humans , Linkage Disequilibrium , Longitudinal Studies , Male , Phenotype , Polymorphism, Single Nucleotide , Pregnancy , Promoter Regions, Genetic , Receptors, Androgen/genetics , Smoking , T-Box Domain Proteins/genetics , Transcription Factor AP-2/genetics , Young AdultABSTRACT
OBJECTIVES: Telomeres are nucleoprotein complexes that cap the ends of linear chromosomes. Telomeric DNA decreases with age and shows considerable heterogeneity in the wider population. There is interest in the application of telomere length measures as a biomarker of general health or "biological age," and the possibility of using mean telomere length to gauge individual disease risk, and to promote lifestyle changes to improve health. This study examined the effectiveness of telomere length as a biomarker for an individual's current overall health status by assessing several measures of general health including SF-36v2 score, current smoking status and a comprehensive obesity phenotype. METHODS: Participants were from the Canterbury Health, Ageing and Lifecourse (CHALICE) cohort, a New Zealand population based multidisciplinary study of aging. Telomere length measurements were obtained on DNA from peripheral blood samples at age 49-51 (n = 351), using a quantitative polymerase chain reaction assay. RESULTS: No associations were found between telomere length measured at age 49-51 and any measures of current health status. The only significant association observed was between telomere length and gender, with females having longer telomere length than men. CONCLUSIONS: Our results suggest that telomere length measurements are unlikely to provide information of much predictive significance for an individual's health status.
Subject(s)
Health Status Indicators , Telomere/physiology , Biomarkers/analysis , Female , Health Status , Humans , Male , Middle Aged , New Zealand , Obesity/physiopathology , Phenotype , Sex FactorsABSTRACT
OBJECTIVES: Anorexia nervosa is a severe psychiatric disorder with high mortality rates. While its aetiology is poorly understood, there is evidence of a significant genetic component. The Anorexia Nervosa Genetics Initiative is an international collaboration which aims to understand the genetic basis of the disorder. This paper describes the recruitment and characteristics of the Australasian Anorexia Nervosa Genetics Initiative sample, the largest sample of individuals with anorexia nervosa ever assembled across Australia and New Zealand. METHODS: Participants completed an online questionnaire based on the Structured Clinical Interview Diagnostic and Statistical Manual of Mental Disorders (4th ed.; DSM-IV) eating disorders section. Participants who met specified case criteria for lifetime anorexia nervosa were requested to provide a DNA sample for genetic analysis. RESULTS: Overall, the study recruited 3414 Australians and 543 New Zealanders meeting the lifetime anorexia nervosa case criteria by using a variety of conventional and social media recruitment methods. At the time of questionnaire completion, 28% had a body mass index ⩽ 18.5 kg/m2. Fasting and exercise were the most commonly employed methods of weight control, and were associated with the youngest reported ages of onset. At the time of the study, 32% of participants meeting lifetime anorexia nervosa case criteria were under the care of a medical practitioner; those with current body mass index < 18.5 kg/m2 were more likely to be currently receiving medical care (56%) than those with current body mass index ⩾ 18.5 kg/m2 (23%). Professional treatment for eating disorders was most likely to have been received from general practitioners (45% of study participants), dietitians (42%) and outpatient programmes (42%). CONCLUSIONS: This study was effective in assembling the largest community sample of people with lifetime anorexia nervosa in Australia and New Zealand to date. The proportion of people with anorexia nervosa currently receiving medical care, and the most common sources of treatment accessed, indicates the importance of training for general practitioners and dietitians in treating anorexia nervosa.
Subject(s)
Anorexia Nervosa/genetics , Patient Selection , Adolescent , Adult , Australia , Body Mass Index , Female , Genome-Wide Association Study , Humans , International Cooperation , Male , New Zealand , Young AdultABSTRACT
We previously used a single nucleotide polymorphism (SNP) in the CHRNA5-A3-B4 gene cluster associated with heaviness of smoking within smokers to confirm the causal effect of smoking in reducing body mass index (BMI) in a Mendelian randomisation analysis. While seeking to extend these findings in a larger sample we found that this SNP is associated with 0.74% lower body mass index (BMI) per minor allele in current smokers (95% CI -0.97 to -0.51, P = 2.00 × 10(-10)), but also unexpectedly found that it was associated with 0.35% higher BMI in never smokers (95% CI +0.18 to +0.52, P = 6.38 × 10(-5)). An interaction test confirmed that these estimates differed from each other (P = 4.95 × 10(-13)). This difference in effects suggests the variant influences BMI both via pathways unrelated to smoking, and via the weight-reducing effects of smoking. It would therefore be essentially undetectable in an unstratified genome-wide association study of BMI, given the opposite association with BMI in never and current smokers. This demonstrates that novel associations may be obscured by hidden population sub-structure. Stratification on well-characterized environmental factors known to impact on health outcomes may therefore reveal novel genetic associations.
Subject(s)
Body Mass Index , Nerve Tissue Proteins/genetics , Receptors, Nicotinic/genetics , Smoking/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Genome-Wide Association Study , Genotype , Health Status , Humans , Middle Aged , Multigene Family , Polymorphism, Single Nucleotide , Severity of Illness Index , Smoking/epidemiology , Weight Loss/genetics , Young AdultABSTRACT
Interest in exploring G-quadruplex (G4) structures in nucleic acids is growing as it becomes more widely recognized that these structures have many interesting biological roles and chemical properties. Probing the G4-forming potential of DNA with dimethyl sulfate, polymerase stop assays, or nuclease digestion are three commonly used techniques that usually employ radio-isotopic labels for visualization. However, as fluorescent labeling methods have grown in popularity and versatility, many laboratories have moved away from the routine use of radio-isotopic methods. We have adapted traditional procedures for structural analysis of G4-forming DNA sequences by using fluorescent labels and capillary electrophoresis and demonstrate their application to well-studied G4 structures, including c-MYC PU27 G4. The three fluorescent assays described here allow interrogation of G4 structures in double- and single-stranded DNA substrates, using either chemical or enzymatic cleavage. When combined, these techniques can provide valuable information for the investigation of G4 topology and structure, as well as visualizing any structural effects caused by interaction of quadruplexes with the complementary C-rich DNA strand.
Subject(s)
DNA, Single-Stranded/chemistry , DNA/chemistry , Fluorescence , G-Quadruplexes , Proto-Oncogene Proteins c-myc/genetics , Circular Dichroism , DNA/analysis , DNA, Single-Stranded/analysis , Fluorescent Dyes/chemistry , HumansABSTRACT
The New Zealand sea lion (NZSL) is of high conservation concern due to its limited distribution and its declining population size. Historically, it occupied most of coastal New Zealand, but is now restricted to a few coastal sites in southern mainland New Zealand and the sub-Antarctic Islands. NZSLs have experienced a recent reduction in population size due to sealing in the 1900s, which is expected to have resulted in increased inbreeding and a loss of genetic variation, potentially reducing the evolutionary capacity of the species and negatively impacting on its long-term prospects for survival. We used 17 microsatellite loci, previously shown to have cross-species applications in pinnipeds, to determine locus- and population-specific statistics for 1205 NZSLs from 7 consecutive breeding seasons. We show that the NZSL population has a moderate level of genetic diversity in comparison to other pinnipeds. We provide genetic evidence for a population reduction, likely caused by historical sealing, and a measure of allele sharing/parental relatedness (internal relatedness) that is suggestive of increased inbreeding in pups that died during recent epizootic episodes. We hypothesize that population bottlenecks and nonrandom mating have impacted on the population genetic architecture of NZSLs, affecting its population recovery.
Subject(s)
Endangered Species , Genetics, Population , Inbreeding , Sea Lions/genetics , Animals , Bayes Theorem , Evolution, Molecular , Genetic Variation , Genotype , Microsatellite Repeats , New ZealandABSTRACT
A functional polymorphism rs1799971 (A118G) in the µ-opioid receptor gene (OPRM1) produces an amino acid substitution Asn40Asp, which is believed to influence naltrexone response in nondepressed alcohol-dependent patients. In this study, patients with alcohol dependence and major depression (n=108) received open-label naltrexone and clinical case management for 12 weeks, and were randomized to citalopram or placebo. General linear mixed models examined the effect of the OPRM1 A118G genotype on alcohol outcomes during treatment. There was no evidence of any difference in the percentage of days abstinent, drinks per drinking day or percentage of heavy drinking days between Asp40 carriers and noncarriers during treatment. This study therefore failed to replicate the previous positive findings for this single nucleotide polymorphism in relation to naltrexone response, possibly indicating that the effect is not present in depressed patients.
Subject(s)
Alcoholism/genetics , Depressive Disorder, Major/genetics , Naltrexone/administration & dosage , Receptors, Opioid, mu/genetics , Alcoholism/complications , Alcoholism/drug therapy , Depressive Disorder, Major/drug therapy , Depressive Disorder, Major/etiology , Female , Genotype , Humans , Male , Naltrexone/pharmacokinetics , Polymorphism, Single NucleotideABSTRACT
DUF1220 protein domains exhibit the greatest human lineage-specific copy number expansion of any protein-coding sequence in the genome, and variation in DUF1220 copy number has been linked to both brain size in humans and brain evolution among primates. Given these findings, we examined associations between DUF1220 subtypes CON1 and CON2 and cognitive aptitude. We identified a linear association between CON2 copy number and cognitive function in two independent populations of European descent. In North American males, an increase in CON2 copy number corresponded with an increase in WISC IQ (R (2) = 0.13, p = 0.02), which may be driven by males aged 6-11 (R (2) = 0.42, p = 0.003). We utilized ddPCR in a subset as a confirmatory measurement. This group had 26-33 copies of CON2 with a mean of 29, and each copy increase of CON2 was associated with a 3.3-point increase in WISC IQ (R (2) = 0.22, p = 0.045). In individuals from New Zealand, an increase in CON2 copy number was associated with an increase in math aptitude ability (R (2) = 0.10 p = 0.018). These were not confounded by brain size. To our knowledge, this is the first study to report a replicated association between copy number of a gene coding sequence and cognitive aptitude. Remarkably, dosage variations involving DUF1220 sequences have now been linked to human brain expansion, autism severity and cognitive aptitude, suggesting that such processes may be genetically and mechanistically inter-related. The findings presented here warrant expanded investigations in larger, well-characterized cohorts.
Subject(s)
Aptitude/physiology , Brain/metabolism , Carrier Proteins/genetics , Chromosomes, Human, Pair 1/genetics , Cognition/physiology , DNA Copy Number Variations/genetics , Intelligence/physiology , Adolescent , Adult , Brain/pathology , Child , Comparative Genomic Hybridization/methods , Female , Follow-Up Studies , Humans , Male , Mathematics , Organ Size , Polymerase Chain Reaction/methods , Protein Structure, Tertiary , Young AdultABSTRACT
The effect of MHC polymorphism on individual fitness variation in the wild remains equivocal; however, much evidence suggests that heterozygote advantage is a major determinant. To understand the contribution of MHC polymorphism to individual disease resistance or susceptibility in natural populations, we investigated two MHC class II B loci, DQB and DRB, in the New Zealand sea lion (NZSL, Phocarctos hookeri). The NZSL is a threatened species which is unusually susceptible to death by bacterial infection at an early age; it has suffered three bacterial induced epizootics resulting in high mortality levels of young pups since 1997. The MHC DQB and DRB haplotypes of dead NZSL pups with known cause of death (bacteria, enteritis or trauma) were sequenced and reconstructed, compared to pups that survived beyond 2 months of age, and distinct MHC DRB allele frequency and genotype differences were identified. Two findings were striking: (i) one DRB allele was present only in dead pups, and (ii) one heterozygous DRB genotype, common in live pups, was absent from dead pups. These results are consistent with some functional relationship with these variants and suggest heterozygote advantage is operating at DRB. We found no association between heterozygosity and fitness at 17 microsatellite loci, indicating that general heterozygosity is not responsible for the effect on fitness detected here. This result may be a consequence of recurrent selection by multiple pathogen assault over recent years and highlights the importance of heterozygote advantage at MHC as a potential mechanism for fitness differences in wild populations.