A Mixture Model Approach in Gene–Gene and Gene–Environmental Interactions for Binary Phenotypes

In translational research, a genetic association study of a binary outcome has a twofold aim: test whether genetic/environmental variables or their combinations are associated with a clinical phenotype, and determine how those combinations are grouped to predict the phenotype (i.e., which combinations have a similarly distributed phenotype, and which ones have differently distributed phenotypes). The second part of this aim has high clinical appeal, because it can directly facilitate clinical decisions. Although traditional logistic regression can detect gene-gene or gene-environmental interaction effects on binary phenotypes, they cannot decisively determine how genotype combinations are grouped to predict the phenotype. Our proposed mixture model approach is valuable in this context. It concurrently detects main and interaction effects of genetic and environmental variables through a likelihood ratio test (LRT) and conducts phenotype cluster analysis based on genetic and environmental variable combinations. The theoretical distribution of the proposed mixture model's likelihood ratio test is robust not only to small sample size but also to unequal sample size in various genotype and environmental subgroups. Hypothesis testing through a likelihood ratio test results in a fast algorithm for p -value calculations. Extensive simulation studies demonstrate that mixture model, overall test in logistic regression, and Monte Carlo based logic regression constantly possess the best power to detect multi-way gene/environmental combinations. The mixture model approach has the highest recovery probability to recover the true partition in the simulation studies. Its applications are exemplified in interim data analyses for two cancer studies.     

Gene–environmental interactions and organophosphate toxicity

Organophosphates (OPs) are an important class of insecticides that in the UK have been widely used for treating sheep for ectoparasites as well as in other sectors of the farming industry. Health problems associated with acute OP toxicity are well defined but, ill-health induced by chronic exposures to OPs remains controversial. A substantial number of sheep farmers complain of chronic ill-health which they attribute to repeated exposure to OPs. If OPs were associated with chronic ill-health then individuals with specific defects in OP metabolism might be expected to be at greater risk of ill-health following exposure. To examine such a hypothesis, the characterisation of both OP exposure and those pathways which lead to the formation and removal of the active OP metabolites becomes important. A wide range of OPs have previously been used to treat sheep but currently the only OP licenced for treating sheep is diazinon. Immediately after treatment, farmers’ urines contain detectable levels of OP metabolites but few farmers have a significant decrease in plasma cholinesterase activity. Diazinon, like chlorpyrifos, is an organothiophosphate which is metabolised, particularly by cytochrome p450s, to the corresponding active oxon form. CYP metabolism also leads to the inactivation of the parent compound and the relative balance of inactivation and activation can depend upon the specific OP and the CYP isoform. OP oxons are inactivated by serum paraoxonase (PON1) and mice lacking PON1 activity are susceptible to oxon and parent OP induced toxicity. PON1 polymorphisms at positions 192 (R form with arginine at 192 and Q with glutamine) and 55 (L form with a leucine and a M form with methionine) influence paroxonase activity. The effect of the Q192R polymorphism is substrate specific with reports indicating that diazoxon is metabolised less by the R isoform. In a study of sheep farmers within the UK, the R allele was associated with an increased risk of self-reported chronic ill-health, a result consistent with the hypothesis that this ill-health may have been caused by OPs. Studies in other populations exposed to pesticides also show associations between ill-health and PON1 Q192R polymorphisms but not consistently so. This is not surprisingly given that exposure is often poorly characterised. In vivo models also suggest that PON1 genotypes may have little influence on susceptibility at low doses of the parent OP. Hence further work is required not only to better characterise OP exposure in humans populations but also to identify those populations susceptible to OP toxicity.     

Gene medicines: the end of the beginning?

First-generation gene medicines and genetic vaccines represent a promising new class of therapeutics that have the potential to prevent, correct, or modulate genetic or acquired diseases. The rational design of synthetic gene delivery and expression systems continues to be essential to enable the precise temporal and spatial control of transgene expression in vivo. With the tantalizing efficacy results and outstanding safety profile observed with nonviral, plasmid-based product candidates in early clinical trials, a multidisciplinary approach remains critical to further improve the effectiveness, reduce the manufacturing costs, and maintain the safety of gene therapeutics and vaccines for their successful development. This commentary provides an historical perspective on somatic gene therapy and briefly addresses the rate-limiting steps in effective gene transfer and expression. The importance of understanding plasmid pharmacokinetics after administration by conventional routes in animal models and in humans is emphasized. Pharmaceutical scientists have a pivotal role to play in deciphering the key biological parameters to effective gene transfer and designing gene delivery systems that will enable plasmid-based products to become an integral part of the future medical armamentarium.     

Gene × environment vulnerability factors for PTSD: the HPA-axis

Post-traumatic stress disorder (PTSD) is a severely debilitating psychiatric condition. Although a lifetime trauma incidence of 40-90% has been reported in the general population, the overall lifetime prevalence for PTSD ranges between 7-12%, suggesting individual-specific differences towards the susceptibility to PTSD. While studies investigating main genetic effects associated with PTSD have yielded inconsistent findings, there is growing evidence supporting the role of gene-environment (G × E) interactions in PTSD. The hypothalamus pituitary adrenal (HPA) axis is one of the main systems activated after exposure to a trauma and perturbations in this system are one of the more consistent neurobiological abnormalities observed in PTSD. Genes regulating the HPA-axis are therefore interesting candidates for G × E studies in PTSD. This article will review the concept and initial results of G × E interactions with polymorphisms in these genes for PTSD. In addition, the use of alternate phenotypes and more complex interaction models such as G × G × E or G × E × E will be explored. Finally, putative molecular mechanisms for these interactions will be presented. The research presented in this article indicates that a combined analysis of environmental, genetic, endophenotype and epigenetic data will be necessary to better understand pathomechanisms in PTSD. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.     

Child μ-Opioid Receptor Gene Variant Influences Parent–Child Relations

Variation in the μ-opioid receptor gene has been associated with early social behavior in mice and rhesus macaques. The current study tested whether the functional OPRM1 A118G predicted various indices of social relations in children. The sample included 226 subjects of self-reported European ancestry (44% female; mean age 13.6, SD=2.2) who were part of a larger representative study of children aged 9–17 years in rural North Carolina. Multiple aspects of recent (past 3 months) parent–child relationship were assessed using the Child and Adolescent Psychiatric Assessment. Parent problems were coded based upon a lifetime history of mental health problems, substance abuse, or criminality. Child genotype interacted with parent behavior such that there were no genotype differences for those with low levels of parent problems; however, when a history of parent problems was reported, the G allele carriers had more enjoyment of parent–child interactions (mean ratio (MR)=3.5, 95% CI=1.6, 8.0) and fewer arguments (MR=3.1, 95% CI=1.1, 8.9). These findings suggest a role for the OPRM1 gene in the genetic architecture of social relations in humans. In summary, a variant in the μ-opioid receptor gene (118G) was associated with improved parent–child relations, but only in the context of a significant disruption in parental functioning.     

Blood–brain Barrier Transport of Non-viral Gene and RNAi Therapeutics

The development of gene- and RNA interference (RNAi)-based therapeutics represents a challenge for the drug delivery field. The global brain distribution of DNA genes, as well as the targeting of specific regions of the brain, is even more complicated because conventional delivery systems, i.e. viruses, have poor diffusion in brain when injected in situ and do not cross the blood–brain barrier (BBB), which is only permeable to lipophilic molecules of less than 400 Da. Recent advances in the “Trojan Horse Liposome” (THL) technology applied to the transvascular non-viral gene therapy of brain disorders presents a promising solution to the DNA/RNAi delivery obstacle. The THL is comprised of immunoliposomes carrying either a gene for protein replacement or small hairpin RNA (shRNA) expression plasmids for RNAi effect, respectively. The THL is engineered with known lipids containing polyethyleneglycol (PEG), which stabilizes its structure in vivo in circulation. The tissue target specificity of THL is given by conjugation of ∼1% of the PEG residues to peptidomimetic monoclonal antibodies (MAb) that bind to specific endogenous receptors (i.e. insulin and transferrin receptors) located on both the BBB and the brain cellular membranes, respectively. These MAbs mediate (a) receptor-mediated transcytosis of the THL complex through the BBB, (b) endocytosis into brain cells and (c) transport to the brain cell nuclear compartment. The present review presents an overview of the THL technology and its current application to gene therapy and RNAi, including experimental models of Parkinson’s disease and brain tumors.     

Temporal Gene expression profile in hippocampus of mouse treated with D-galactose

Rodent chronically treated with D-galactose （D-gal） is emerging as a potential aging model in pharmacological studies. However its mechanism remains unclear. We investigated the gene alterations in hippocampus of D-gal treated mice. C57 mice treated with vehicle or D-gal for 2, 4 and 8 weeks were first subjected to behavioural tests. Both 4 and 8-week D-gal treatment could lead to lower discrimination index in object recognition test, but only 8-week D-gal treated mice showed significant spatial learning ＆ memory impairment in Morris water maze. Gene expression profiles in mouse hippocampus were then examined with cDNA microarray.     

Rapid and Sensitive Assay of Tumor Necrosis Factor-α Gene Transcription

Pharmaceutical Research -     

The Research on Chimeric MHC-Ⅰ Gene Responsing to Allogeneic Dendritic Cells

目的:构建分子嵌合主要组织相容性复合体(MHC)-Ⅰ基因小鼠骨髓造血干细胞,并探讨其诱导脾脏T细胞对异基因小鼠树突状细胞(DC)反应的机制.方法:密度梯度法分离培养BALB/c小鼠骨髓造血干细胞.构建携带C57BL/6小鼠MHC-Ⅰ基因慢病毒载体(病毒感染组),携带无意义基因慢病毒载体(阴性对照组).分别感染BALB/c 小鼠骨髓造血干细胞,构建分子嵌合细胞.分别取病毒感染组、阴性对照组及未加入病毒的空白对照组造血干细胞输注BALB/c小鼠后7d,获取脾脏T淋巴细胞,分别与C57BL/6小鼠DC进行混合淋巴细胞培养,测定刺激指数.结果:成功体外分选及培养BALB/c小鼠骨髓造血干细胞.病毒感染组C57BL/6小鼠MHC-Ⅰ蛋白表达率可达98.17％.单向混合淋巴细胞培养结果显示,C57BL/6小鼠DC对输注病毒感染组细胞后BALB/c小鼠脾脏T细胞刺激指数明显降低(P＜0.01).结论:输注分子嵌合MHC-Ⅰ基因造血干细胞后,小鼠脾脏T细胞对异基因小鼠DC反应明显减低.     

Angiopep-Conjugated Nanoparticles for Targeted Long-Term Gene Therapy of Parkinson’s Disease

Purpose

To prepare an angiopep-conjugated dendrigraft poly-L-lysine (DGL)-based gene delivery system and evaluate the neuroprotective effects in the rotenone-induced chronic model of Parkinson’s disease (PD).

Methods

Angiopep was applied as a ligand specifically binding to low-density lipoprotein receptor-related protein (LRP) which is overexpressed on blood-brain barrier (BBB), and conjugated to biodegradable DGL via hydrophilic polyethyleneglycol (PEG), yielding DGL-PEG-angiopep (DPA). In vitro characterization was carried out. The neuroprotective effects were evaluated in a chronic parkinsonian model induced by rotenone using a regimen of multiple dosing intravenous administrations.

Results

The successful synthesis of DPA was demonstrated via ¹H-NMR. After encapsulating the therapeutic gene encoding human glial cell line-derived neurotrophic factor (hGDNF), DPA/hGDNF NPs showed a sphere-like shape with the size of 119?±?12 nm and zeta potential of 8.2?±?0.7 mV. Angiopep-conjugated NPs exhibited higher cellular uptake and gene expression in brain cells compared to unmodified counterpart. The pharmacodynamic results showed that rats in the group with five injections of DPA/hGDNF NPs obtained best improved locomotor activity and apparent recovery of dopaminergic neurons compared to those in other groups.

Conclusion

This work provides a practical non-viral gene vector for long-term gene therapy of chronic neurodegenerative disorders.     

Gene therapy in psychiatric disorders: too early,too complex?

Gene transfer strategies are being tested in a variety of animal models for psychiatric disorders. These promise to translate viral-mediated, non-viral and combinatorial techniques for delivery of transgenes into neuroanatomical and cell-type-specific therapeutic tools. However, these disorders involve complex functional neurocircuits and developmental aetiologies that may present even greater challenges than other neurological conditions.     

Adenoviral Gene Delivery to Solid Tumors by Recombinant Silk–Elastinlike Protein Polymers

Purpose The purpose of this study was to investigate the potential of silk–elastinlike protein polymers (SELPs) in controlling the release rate of adenoviruses in vitro and in vivo while preserving their bioactivity. Materials and Methods A hydrogel system composed of SELP/adenovirus mixture was prepared. The release of the adenovirus particles from the hydrogels was quantified by Real Time-PCR and the bioactivity of the released viruses was evaluated using confocal microscopy and β-galactosidase assay. To demonstrate the ability of SELP in entrapping virus cargo and releasing it over a prolonged period of time in vivo, a SELP/adenovirus mixture was prepared and injected directly into xenograft tumor models of breast and head and neck cancer in mice. At various time points mice were sacrificed, tumors dissected, and tissue sections studied under confocal microscope. Results In vitro studies demonstrated that SELP hydrogels release viruses over a period of 4 weeks while preserving their bioactivity. After intratumoral injection, a prolonged and localized expression of adenoviruses was observed. Conclusions These results suggest the potential of SELPs in local adenoviral delivery to solid tumors as an alternative approach to intratumoral virus infusion. Electronic supplementary material The online version of this article (doi: contains supplementary material, which is available to authorized users.     

Gene therapy: a strategy for the treatment of inherited muscle diseases?

The emergence of new vectors of viral origin (recombinant adeno-associated viruses, second and third generation adenoviruses) and a new potential source of cells for transplantation (muscle-derived stem cells) are broadening the panel of therapeutic options for myopathies. Although the perfect gene-transfer method(s) have not yet been found, recent findings will certainly constitute a strong knowledge base for future clinical trials.     

Gene expression and antitumor effect following imelectroporation delivery of human interferon α2 gene

AIM: To investigate the gene expression and antitumor effect following im electroporation delivery of human interferon alpha 2 (hIFN-alpha 2) gene. METHODS: The pcD2/hIFN-alpha 2 was injected into the middle of the quadriceps muscle of female BALB/c mice or the leukemia-bearing female BALB/c nude mice, and then electroporation was given to the injection site. Optimal electrical parameters and the efficiency of gene transfer was studied with hIFN-alpha 2 ELISA kit. The HL-60 tumor model in BALB/c nude mice was used to investigate therapeutic effects of im electroporation delivery of pcD2/hIFN-alpha 2. RESULTS: The optimal conditions for the electric pulses were as follows: voltage at 200 V/cm; pulse duration at 40 ms per pulse; number of pulse at 6 pulses and frequency at 1 Hz. Under optimal conditions, the serum hIFN-alpha 2 levels in electroporation group (160 microg/L+/-31 microg/L) were 45-fold higher than those of nonelectroporation group (3.6 microg/L+/-1.6 microg/L, P<0.01). The growth of leukemia was inhibited more obviously and the survival time of the leukemia-bearing nude mice was prolonged after im electroporation delivery of pcD2/hIFN-alpha 2 100 microg or 200 microg. CONCLUSION: Electroporation was an efficient method for the delivery of plasmid DNA and im electroporation delivery of pcD2/hIFN-alpha 2 was effective in treating leukemia.