Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

The effect of BCMO1 gene variants on macular pigment optical density in young healthy Caucasians

Background Serum lutein (L) and zeaxanthin (Z) positively correlate with macular pigment optical density (MPOD), hence the latter is a valuable indirect tool for measuring L and Z content in the macula. L and Z have been attributed antioxidant capacity and protection from certain retinal diseases but their uptake within the eye is thought to depend on genetic, age and environmental factors. In particular gene variants within beta-carotene monooxygenase (BCMO1) are thought to modulate MPOD in the macula. Objectives: To determine the effect of BCMO1 single nucleotide polymorphisms (SNPs) rs11645428, rs6420424 and rs6464851 on macular pigment optical density (MPOD) in a cohort of young healthy participants of Caucasian origin with normal ocular health. Design In this cohort study, MPOD was assessed in 46 healthy participants (22 male and 24 female) with a mean age of 24 ± 4.0 years (range 19-33). The three SNPs, rs11645428, rs6420424, rs6564851 that have established associations with MPOD were determined using MassEXTEND (hME) Sequenom assay. One-way analysis of variance (ANOVA) was performed on groups segregated into homozygous and heterozygous BCMO1 genotypes. Correlations between body mass index (BMI), iris colour, gender, central retinal thickness (CRT), diet and MPOD were investigated. Results MPOD did not significantly vary with BCMO1 rs11645428 (F2,41 = 0.700, p = 0.503), rs6420424 (F2,41 = 0.210, p = 0.801) nor rs6464851 homozygous or heterozygous genotypes (F2,41 = 0,13, p = 0.88), in this young healthy cohort. The combination of these three SNPs into triple genotypes based on plasma conversion efficiency did not affect MPOD (F2,41 = 0.07, p = 0.9). There was a significant negative correlation with MPOD and central retinal thickness (r = - 0.39, p = 0.01) but no significant correlation between BMI, iris colour, gender and MPOD. Conclusion Our results indicate that macular pigment deposition within the central retina is not dependent on BCMO1 gene variants in young healthy people. We propose that MPOD is saturated in younger persons and/or other gene variant combinations determine its deposition.

Relationship of a variant in the NTRK1 gene to white matter microstructure in young adults

The NTRK1 gene (also known as TRKA) encodes a high-affinity receptor for NGF, a neurotrophin involved in nervous system development and myelination. NTRK1 has been implicated in neurological function via links between the T allele at rs6336 (NTRK1-T) and schizophrenia risk. A variant in the neurotrophin gene, BDNF, was previously associated with white matter integrity in young adults, highlighting the importantce of neurotrophins to white matter development. We hypothesized that NTRK1-T would relate to lower fractional anisotropy in healthy adults. We scanned 391 healthy adult human twins and their siblings (mean age: 23.6 ± 2.2 years; 31 NTRK1-T carriers, 360 non-carriers) using 105-gradient diffusion tensor imaging at 4 tesla. We evaluated in brain white matter how NTRK1-T and NTRK1 rs4661063 allele A (rs4661063-A, which is in moderate linkage disequilibrium with rs6336) related to voxelwise fractional anisotropy-acommondiffusion tensor imaging measure of white matter microstructure. We used mixed-model regression to control for family relatedness, age, and sex. The sample was split in half to test reproducibility of results. The false discovery rate method corrected for voxelwise multiple comparisons. NTRK1-T and rs4661063-A correlated with lower white matter fractional anisotropy, independent of age and sex (multiple-comparisons corrected: false discovery rate critical p=0.038 forNTRK1-Tand0.013 for rs4661063-A). In each half-sample, theNTRK1-T effectwasreplicated in the cingulum, corpus callosum, superior and inferior longitudinal fasciculi, inferior fronto-occipital fasciculus, superior corona radiata, and uncinate fasciculus. Our results suggest that NTRK1-T is important for developing white matter microstructure.

Common Alzheimer's disease risk variant within the CLU gene affects white matter microstructure in young adults

There is a strong genetic risk for late-onset Alzheimer's disease (AD), but so far few gene variants have been identified that reliably contribute to that risk. A newly confirmed genetic risk allele C of the clusterin (CLU) gene variant rs11136000 is carried by ~88% of Caucasians. The C allele confers a 1.16 greater odds of developing late-onset AD than the T allele. AD patients have reductions in regional white matter integrity. We evaluated whether the CLU risk variant was similarly associated with lower white matter integrity in healthy young humans. Evidence of early brain differences would offer a target for intervention decades before symptom onset. We scanned 398 healthy young adults (mean age, 23.6 ± 2.2 years) with diffusion tensor imaging, a variation of magnetic resonance imaging sensitive to white matter integrity in the living brain. We assessed genetic associations using mixed-model regression at each point in the brain to map the profile of these associations with white matter integrity. Each C allele copy of the CLUvariant was associated with lower fractional anisotropy-a widely accepted measure of white matter integrity-in multiple brain regions, including several known to degenerate in AD. These regions included the splenium of the corpus callosum, the fornix, cingulum, and superior and inferior longitudinal fasciculi in both brain hemispheres. Young healthy carriers of the CLU gene risk variant showed a distinct profile of lower white matter integrity that may increase vulnerability to developing AD later in life.

Relation between variants in the neurotrophin receptor gene, NTRK3, and white matter integrity in healthy young adults

The NTRK3 gene (also known as TRKC) encodes a high affinity receptor for the neurotrophin 3'-nucleotidase (NT3), which is implicated in oligodendrocyte and myelin development. We previously found that white matter integrity in young adults is related to common variants in genes encoding neurotrophins and their receptors. This underscores the importance of neurotrophins for white matter development. NTRK3 variants are putative risk factors for schizophrenia, bipolar disorder, and obsessive-compulsive disorder hoarding, suggesting that some NTRK3 variants may affect the brain.To test this, we scanned 392 healthy adult twins and their siblings (mean age, 23.6. ±. 2.2. years; range: 20-29. years) with 105-gradient 4-Tesla diffusion tensor imaging (DTI). We identified 18 single nucleotide polymorphisms (SNPs) in the NTRK3 gene that have been associated with neuropsychiatric disorders. We used a multi-SNP model, adjusting for family relatedness, age, and sex, to relate these variants to voxelwise fractional anisotropy (FA) - a DTI measure of white matter integrity.FA was optimally predicted (based on the highest false discovery rate critical p), by five SNPs (rs1017412, rs2114252, rs16941261, rs3784406, and rs7176429; overall FDR critical p=. 0.028). Gene effects were widespread and included the corpus callosum genu and inferior longitudinal fasciculus - regions implicated in several neuropsychiatric disorders and previously associated with other neurotrophin-related genetic variants in an overlapping sample of subjects. NTRK3 genetic variants, and neurotrophins more generally, may influence white matter integrity in brain regions implicated in neuropsychiatric disorders.

Extending genetic linkage analysis to diffusion tensor images to map single gene effects on brain fiber architecture

We extended genetic linkage analysis - an analysis widely used in quantitative genetics - to 3D images to analyze single gene effects on brain fiber architecture. We collected 4 Tesla diffusion tensor images (DTI) and genotype data from 258 healthy adult twins and their non-twin siblings. After high-dimensional fluid registration, at each voxel we estimated the genetic linkage between the single nucleotide polymorphism (SNP), Val66Met (dbSNP number rs6265), of the BDNF gene (brain-derived neurotrophic factor) with fractional anisotropy (FA) derived from each subject's DTI scan, by fitting structural equation models (SEM) from quantitative genetics. We also examined how image filtering affects the effect sizes for genetic linkage by examining how the overall significance of voxelwise effects varied with respect to full width at half maximum (FWHM) of the Gaussian smoothing applied to the FA images. Raw FA maps with no smoothing yielded the greatest sensitivity to detect gene effects, when corrected for multiple comparisons using the false discovery rate (FDR) procedure. The BDNF polymorphism significantly contributed to the variation in FA in the posterior cingulate gyrus, where it accounted for around 90-95% of the total variance in FA. Our study generated the first maps to visualize the effect of the BDNF gene on brain fiber integrity, suggesting that common genetic variants may strongly determine white matter integrity.

Hierarchical clustering of the genetic connectivity matrix reveals the network topology of gene action on brain microstructure: An N=531 twin study

Genetic correlation (rg) analysis determines how much of the correlation between two measures is due to common genetic influences. In an analysis of 4 Tesla diffusion tensor images (DTI) from 531 healthy young adult twins and their siblings, we generalized the concept of genetic correlation to determine common genetic influences on white matter integrity, measured by fractional anisotropy (FA), at all points of the brain, yielding an NxN genetic correlation matrix rg(x,y) between FA values at all pairs of voxels in the brain. With hierarchical clustering, we identified brain regions with relatively homogeneous genetic determinants, to boost the power to identify causal single nucleotide polymorphisms (SNP). We applied genome-wide association (GWA) to assess associations between 529,497 SNPs and FA in clusters defined by hubs of the clustered genetic correlation matrix. We identified a network of genes, with a scale-free topology, that influences white matter integrity over multiple brain regions.

Gene network effects on brain microstructure and intellectual performance identified in 472 twins

A major challenge in neuroscience is finding which genes affect brain integrity, connectivity, and intellectual function. Discovering influential genes holds vast promise for neuroscience, but typical genome-wide searches assess approximately one million genetic variants one-by-one, leading to intractable false positive rates, even with vast samples of subjects. Even more intractable is the question of which genes interact and how they work together to affect brain connectivity. Here, we report a novel approach that discovers which genes contribute to brain wiring and fiber integrity at all pairs of points in a brain scan. We studied genetic correlations between thousands of points in human brain images from 472 twins and their nontwin siblings (mean age: 23.7 2.1 SD years; 193 male/279 female).Wecombined clustering with genome-wide scanning to find brain systems withcommongenetic determination.Wethen filtered the image in a new way to boost power to find causal genes. Using network analysis, we found a network of genes that affect brain wiring in healthy young adults. Our new strategy makes it computationally more tractable to discover genes that affect brain integrity. The gene network showed small-world and scale-free topologies, suggesting efficiency in genetic interactions and resilience to network disruption. Genetic variants at hubs of the network influence intellectual performance by modulating associations between performance intelligence quotient and the integrity of major white matter tracts, such as the callosal genu and splenium, cingulum, optic radiations, and the superior longitudinal fasciculus.

BDNF gene effects on brain circuitry replicated in 455 twins

Brain-derived neurotrophic factor (BDNF) plays a key role in learning and memory, but its effects on the fiber architecture of the living brain are unknown. We genotyped 455 healthy adult twins and their non-twin siblings (188 males/267 females; age: 23.7 ± 2.1. years, mean ± SD) and scanned them with high angular resolution diffusion tensor imaging (DTI), to assess how the BDNF Val66Met polymorphism affects white matter microstructure. By applying genetic association analysis to every 3D point in the brain images, we found that the Val-BDNF genetic variant was associated with lower white matter integrity in the splenium of the corpus callosum, left optic radiation, inferior fronto-occipital fasciculus, and superior corona radiata. Normal BDNF variation influenced the association between subjects' performance intellectual ability (as measured by Object Assembly subtest) and fiber integrity (as measured by fractional anisotropy; FA) in the callosal splenium, and pons. BDNF gene may affect the intellectual performance by modulating the white matter development. This combination of genetic association analysis and large-scale diffusion imaging directly relates a specific gene to the fiber microstructure of the living brain and to human intelligence.

Obesity gene NEGR1 associated with white matter integrity in healthy young adults

Obesity is a crucial public health issue in developed countries, with implications for cardiovascular and brain health as we age. A number of commonly-carried genetic variants are associated with obesity. Here we aim to see whether variants in obesity-associated genes - NEGR1, FTO, MTCH2, MC4R, LRRN6C, MAP2K5, FAIM2, SEC16B, ETV5, BDNF- AS, ATXN2L, ATP2A1, KCTD15, and TNN13K - are associated with white matter microstructural properties, assessed by high angular resolution diffusion imaging (HARDI) in young healthy adults between 20 and 30. years of age from the Queensland Twin Imaging study (QTIM). We began with a multi-locus approach testing how a number of common genetic risk factors for obesity at the single nucleotide polymorphism (SNP) level may jointly influence white matter integrity throughout the brain and found a wide spread genetic effect. Risk allele rs2815752 in NEGR1 was most associated with lower white matter integrity across a substantial portion of the brain. Across the area of significance in the bilateral posterior corona radiata, each additional copy of the risk allele was associated with a 2.2% lower average FA. This is the first study to find an association between an obesity risk gene and differences in white matter integrity. As our subjects were young and healthy, our results suggest that NEGR1 has effects on brain structure independent of its effect on obesity.

Alzheimer's disease risk gene, GAB2, is associated with regional brain volume differences in 755 young healthy twins

The development of late-onset Alzheimer's disease (LOAD) is under strong genetic control and there is great interest in the genetic variants that confer increased risk. The Alzheimer's disease risk gene, growth factor receptor bound protein 2-associated protein (GAB2), has been shown to provide a 1.27- 1.51 increased odds of developing LOAD for rs7101429 major allele carriers, in case-control analysis. GAB2 is expressed across the brain throughout life, and its role in LOAD pathology is well understood. Recent studies have begun to examine the effect of genetic variation in the GAB2 gene on differences in the brain. However, the effect of GAB2 on the young adult brain has yet to be considered. Here we found a significant association between the GAB2 gene and morphological brain differences in 755 young adult twins (469 females) (M = 23.1, SD = 3.1 years), using a gene-based test with principal components regression (PCReg). Detectable differences in brain morphology are therefore associated with variation in the GAB2 gene, even in young adults, long before the typical age of onset of Alzheimer's disease.

Brain structure in healthy adults is related to serum transferrin and the H63D polymorphism in the HFE gene

Control of iron homeostasis is essential for healthy central nervous system function: iron deficiency is associated with cognitive impairment, yet iron overload is thought to promote neurodegenerative diseases. Specific genetic markers have been previously identified that influence levels of transferrin, the protein that transports iron throughout the body, in the blood and brain. Here, we discovered that transferrin levels are related to detectable differences in the macro- and microstructure of the living brain. We collected brain MRI scans from 615 healthy young adult twins and siblings, of whom 574 were also scanned with diffusion tensor imaging at 4 Tesla. Fiber integrity was assessed by using the diffusion tensor imaging-based measure of fractional anisotropy. In bivariate genetic models based on monozygotic and dizygotic twins, we discovered that partially overlapping additive genetic factors influenced transferrin levels and brain microstructure. We also examined common variants in genes associated with transferrin levels, TF and HFE, and found that a commonly carried polymorphism (H63D at rs1799945) in the hemochromatotic HFE gene was associated with white matter fiber integrity. This gene has a well documented association with iron overload. Our statistical maps reveal previously unknown influences of the same gene on brain microstructure and transferrin levels. This discovery may shed light on the neural mechanisms by which iron affects cognition, neurodevelopment, and neurodegeneration.

Genome-wide scan of healthy human connectome discovers SPON1 gene variant influencing dementia severity

Aberrant connectivity is implicated in many neurological and psychiatric disorders, including Alzheimer's disease and schizophrenia. However, other than a few disease-associated candidate genes, we know little about the degree to which genetics play a role in the brain networks; we know even less about specific genes that influence brain connections. Twin and family-based studies can generate estimates of overall genetic influences on a trait, but genome-wide association scans (GWASs) can screen the genome for specific variants influencing the brain or risk for disease. To identify the heritability of various brain connections, we scanned healthy young adult twins with high-field, highangular resolution diffusion MRI. We adapted GWASs to screen the brain's connectivity pattern, allowing us to discover genetic variants that affect the human brain's wiring. The association of connectivity with the SPON1 variant at rs2618516 on chromosome 11 (11p15.2) reached connectome-wide, genome-wide significance after stringent statistical corrections were enforced, and it was replicated in an independent subsample. rs2618516 was shown to affect brain structure in an elderly population with varying degrees of dementia. Older people who carried the connectivity variant had significantly milder clinical dementia scores and lower risk of Alzheimer's disease. As a posthoc analysis, we conducted GWASs on several organizational and topological network measures derived from the matrices to discover variants in and around genes associated with autism (MACROD2), development (NEDD4), and mental retardation (UBE2A) significantly associated with connectivity. Connectome-wide, genome-wide screening offers substantial promise to discover genes affecting brain connectivity and risk for brain diseases.

Discovery and replication of gene influences on brain structure using LASSO regression

We implemented least absolute shrinkage and selection operator (LASSO) regression to evaluate gene effects in genome-wide association studies (GWAS) of brain images, using an MRI-derived temporal lobe volume measure from 729 subjects scanned as part of the Alzheimer's Disease Neuroimaging Initiative (ADNI). Sparse groups of SNPs in individual genes were selected by LASSO, which identifies efficient sets of variants influencing the data. These SNPs were considered jointly when assessing their association with neuroimaging measures. We discovered 22 genes that passed genome-wide significance for influencing temporal lobe volume. This was a substantially greater number of significant genes compared to those found with standard, univariate GWAS. These top genes are all expressed in the brain and include genes previously related to brain function or neuropsychiatric disorders such as MACROD2, SORCS2, GRIN2B, MAGI2, NPAS3, CLSTN2, GABRG3, NRXN3, PRKAG2, GAS7, RBFOX1, ADARB2, CHD4, and CDH13. The top genes we identified with this method also displayed significant and widespread post hoc effects on voxelwise, tensor-based morphometry (TBM) maps of the temporal lobes. The most significantly associated gene was an autism susceptibility gene known as MACROD2.We were able to successfully replicate the effect of the MACROD2 gene in an independent cohort of 564 young, Australian healthy adult twins and siblings scanned with MRI (mean age: 23.8±2.2 SD years). Our approach powerfully complements univariate techniques in detecting influences of genes on the living brain.

A commonly carried genetic variant in the delta opioid receptor gene, OPRD1, is associated with smaller regional brain volumes: Replication in elderly and young populations

Delta opioid receptors are implicated in a variety of psychiatric and neurological disorders. These receptors play a key role in the reinforcing properties of drugs of abuse, and polymorphisms in OPRD1 (the gene encoding delta opioid receptors) are associated with drug addiction. Delta opioid receptors are also involved in protecting neurons against hypoxic and ischemic stress. Here, we first examined a large sample of 738 elderly participants with neuroimaging and genetic data from the Alzheimer's Disease Neuroimaging Initiative. We hypothesized that common variants in OPRD1 would be associated with differences in brain structure, particularly in regions relevant to addictive and neurodegenerative disorders. One very common variant (rs678849) predicted differences in regional brain volumes. We replicated the association of this single-nucleotide polymorphism with regional tissue volumes in a large sample of young participants in the Queensland Twin Imaging study. Although the same allele was associated with reduced volumes in both cohorts, the brain regions affected differed between the two samples. In healthy elderly, exploratory analyses suggested that the genotype associated with reduced brain volumes in both cohorts may also predict cerebrospinal fluid levels of neurodegenerative biomarkers, but this requires confirmation. If opiate receptor genetic variants are related to individual differences in brain structure, genotyping of these variants may be helpful when designing clinical trials targeting delta opioid receptors to treat neurological disorders.