Cognitive Decline in Alzheimer's Disease: Limited Clinical Utility for GWAS or Polygenic Risk Scores in a Clinical Trial Setting.

Introduction: Alzheimer's disease (AD) is a progressive and irreversible neurological disease. The genetics and molecular mechanisms underpinning differential cognitive decline in AD are not well understood; the genetics of AD risk have been studied far more assiduously. Materials and Methods: Two phase III clinical trials measuring cognitive decline over 48 weeks using Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog, n = 2060) and Clinical Dementia Rating-Sum of Boxes (CDR-SB, n = 1996) were retrospectively genotyped. A Genome-Wide Association Study (GWAS) was performed to identify and replicate genetic variants associated with cognitive decline. The relationship between polygenic risk score (PRS) and cognitive decline was tested to investigate the predictive power of aggregating many variants of individually small effect. Results: No loci met candidate gene or genome-wide significance. PRS explained a very small percentage of variance in rates of cognitive decline (ADAS-cog: 0.54%). Conclusions: These results suggest that incorporating genetic information in the prediction of cognitive decline in AD currently appears to have limited utility in clinical trials, consistent with small effect sizes estimated elsewhere. If AD progression is more heritable soon after disease onset, genetics may have more clinical utility.

Protection of Mice from Lethal Vaccinia Virus Infection by Vaccinia Virus Protein Subunits with a CpG Adjuvant.

Smallpox vaccination carries a high risk of adverse events in recipients with a variety of contra-indications for live vaccines. Although alternative non-replicating vaccines have been described in the form of replication-deficient vaccine viruses, DNA vaccines, and subunit vaccines, these are less efficacious than replicating vaccines in animal models. DNA and subunit vaccines in particular have not been shown to give equivalent protection to the traditional replicating smallpox vaccine. We show here that combinations of the orthopoxvirus A27, A33, B5 and L1 proteins give differing levels of protection when administered in different combinations with different adjuvants. In particular, the combination of B5 and A27 proteins adjuvanted with CpG oligodeoxynucleotides (ODN) gives a level of protection in mice that is equivalent to the Lister traditional vaccine in a lethal vaccinia virus challenge model.

Communicating BRCA research results to patients enrolled in international clinical trials: lessons learnt from the AGO-OVAR 16 study.

BACKGROUND: The focus on translational research in clinical trials has the potential to generate clinically relevant genetic data that could have importance to patients. This raises challenging questions about communicating relevant genetic research results to individual patients. METHODS: An exploratory pharmacogenetic analysis was conducted in the international ovarian cancer phase III trial, AGO-OVAR 16, which found that patients with clinically important germ-line BRCA1/2 mutations had improved progression-free survival prognosis. Mechanisms to communicate BRCA results were evaluated, because these findings may be beneficial to patients and their families. RESULTS: Communicating individual BRCA results was not anticipated during clinical trial design. Consequently, options were not available for patients to indicate their preference for receiving their individual results when they signed pharmacogenetic informed consent. Differences in local requirements, clinical practice, and opinion regarding the ethical aspects of how to convey genetic results to patients are all potential barriers to returning individual BRCA results to patients. Communicating the aggregate BRCA result from this study provided clinical investigators with a mechanism to disseminate the overall study finding to patients while taking individual circumstances, local guidelines and clinical practice into account. CONCLUSION: This study illustrates the importance of increasing the clarity and scope of informed consent and the need for patient engagement to ensure clinical trial participants can indicate their preference regarding receipt of potentially important individual pharmacogenetic results. TRIAL REGISTRATION: This study was registered in the NCT Clinical Trial Registry under NCT00866697 on March 19, 2009, following approval from participating ethics committees (Additional file 1).

Comparison of the performance of three PCR assays for the detection and differentiation of Theileria orientalis genotypes.

BACKGROUND: Oriental theileriosis is a tick-borne disease of bovines caused by the members of the Theileria orientalis complex. Recently, we developed a multiplexed tandem (MT) PCR to detect, differentiate and quantitate four genotypes (i.e., buffeli, chitose, ikeda and type 5) of T. orientalis. In this study, we used MT PCR to assess the prevalence and infection intensity of four T. orientalis genotypes in selected cattle herds that experienced oriental theileriosis outbreaks in New Zealand, and compared the sensitivities and specificities of MT PCR, PCR-high resolution melting (PCR-HRM) and a TaqMan qPCR. METHODS: MT PCR, PCR-HRM analysis for T. orientalis and a TaqMan qPCR assay for ikeda genotype were employed to test 154 and 88 cattle blood samples from North (where oriental theileriosis outbreaks had occurred; designated as Group 1) and South (where no outbreaks had been reported; Group 2) Islands of New Zealand, respectively. Quantitative data from MT PCR assay were analyzed using generalized linear model and paired-sample t-test. The diagnostic specificity and sensitivity of the assays were estimated using a Bayesian latent class modeling approach. RESULTS: In Group 1, 99.4% (153/154) of cattle were test-positive for T. orientalis in both the MT PCR and PCR-HRM assays. The apparent prevalences of genotype ikeda in Group 1 were 87.6% (134/153) and 87.7% (135/154) using the MT PCR and Ikeda TaqMan qPCR assays, respectively. Using the MT PCR test, all four genotypes of T. orientalis were detected. The infection intensity estimated for genotype ikeda was significantly higher (P = 0.009) in severely anaemic cattle than in those without anaemia, and this intensity was significantly higher than that of buffeli (P < 0.001) in the former cattle. Bayesian latent class analysis showed that the diagnostic sensitivities (97.1-98.9%) and specificities (96.5-98.9%) of the three PCR assays were very comparable. CONCLUSION: The present findings show the advantages of using the MT PCR assay as a useful tool for in-depth epidemiological and transmission studies of T. orientalis worldwide.

An update to returning genetic research results to individuals: perspectives of the industry pharmacogenomics working group.

The ease with which genotyping technologies generate tremendous amounts of data on research participants has been well chronicled, a feat that continues to become both faster and cheaper to perform. In parallel to these advances come additional ethical considerations and debates, one of which centers on providing individual research results and incidental findings back to research participants taking part in genetic research efforts. In 2006 the Industry Pharmacogenomics Working Group (I-PWG) offered some 'Points-to-Consider' on this topic within the context of the drug development process from those who are affiliated to pharmaceutical companies. Today many of these points remain applicable to the discussion but will be expanded upon in this updated viewpoint from the I-PWG. The exploratory nature of pharmacogenomic work in the pharmaceutical industry is discussed to provide context for why these results typically are not best suited for return. Operational challenges unique to this industry which cause barriers to returning this information are also explained.

Saliva sampling in global clinical studies: the impact of low sampling volume on performance of DNA in downstream genotyping experiments.

BACKGROUND: The collection of viable DNA samples is an essential element of any genetics research programme. Biological samples for DNA purification are now routinely collected in many studies with a variety of sampling methods available. Initial observation in this study suggested a reduced genotyping success rate of some saliva derived DNA samples when compared to blood derived DNA samples prompting further investigation. METHODS: Genotyping success rate was investigated to assess the suitability of using saliva samples in future safety and efficacy pharmacogenetics experiments. The Oragene® OG-300 DNA Self-Collection kit was used to collect and extract DNA from saliva from 1468 subjects enrolled in global clinical studies. Statistical analysis evaluated the impact of saliva sample volume of collection on the quality, yield, concentration and performance of saliva DNA in genotyping assays. RESULTS: Across 13 global clinical studies that utilized the Oragene® OG-300 DNA Self-Collection kit there was variability in the volume of saliva sample collection with ~31% of participants providing 0.5 mL of saliva, rather than the recommended 2 mL. While the majority of saliva DNA samples provided high quality genotype data, collection of 0.5 mL volumes of saliva contributed to DNA samples being significantly less likely to pass genotyping quality control standards. Assessment of DNA sample characteristics that may influence genotyping outcomes indicated that saliva sample volume, DNA purity and turbidity were independently associated with sample genotype pass rate, but that saliva collection volume had the greatest effect. CONCLUSION: When employing saliva sampling to obtain DNA, it is important to encourage all study participants to provide sufficient sample to minimize potential loss of data in downstream genotyping experiments.

An 18-kDa translocator protein (TSPO) polymorphism explains differences in binding affinity of the PET radioligand PBR28.

[(11)C]PBR28 binds the 18-kDa Translocator Protein (TSPO) and is used in positron emission tomography (PET) to detect microglial activation. However, quantitative interpretations of signal are confounded by large interindividual variability in binding affinity, which displays a trimodal distribution compatible with a codominant genetic trait. Here, we tested directly for an underlying genetic mechanism to explain this. Binding affinity of PBR28 was measured in platelets isolated from 41 human subjects and tested for association with polymorphisms in TSPO and genes encoding other proteins in the TSPO complex. Complete agreement was observed between the TSPO Ala147Thr genotype and PBR28 binding affinity phenotype (P value=3.1 × 10(-13)). The TSPO Ala147Thr polymorphism predicts PBR28 binding affinity in human platelets. As all second-generation TSPO PET radioligands tested hitherto display a trimodal distribution in binding affinity analogous to PBR28, testing for this polymorphism may allow quantitative interpretation of TSPO PET studies with these radioligands.

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus.

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.

Chronic lithium administration down regulates transthyretin mRNA expression in rat choroid plexus.

Transthyretin (TTR) accounts for a quarter of the protein content of ventricular cerebrospinal fluid (CSF) yet its exact role in the brain remains unknown. Patients with a diagnosis of depression have reduced CSF levels of TTR and the locus encoding the TTR gene has been implicated in a Danish pedigree of bipolar patients. Lithium, the major treatment for bipolar disorder in the UK, was subcutaneously infused into rats for 28 days in the form of lithium chloride using osmotic minipumps. In situ hybridizations using oligonucleotide probes targeted against the TTR transcript were performed on coronal brain sections. Lithium significantly reduced the level of transthyretin mRNA in the rat choroid plexus within the lateral and third ventricle. The down-regulation was confirmed using semi-quantitative reverse transcription PCR on dissected brain tissue. Recent studies in mice suggest that the TTR gene is implicated in depression-like behavior therefore this effect of lithium may be relevant to its use as a mood stabilizer or an adjuvant to antidepressant drugs.

Gene dosage effect on gamma-secretase component Aph-1b in a rat model for neurodevelopmental disorders.

A combination of genetic factors and early life events is thought to determine the vulnerability of an individual to develop a complex neurodevelopmental disorder like schizophrenia. Pharmacogenetically selected, apomorphine-susceptible Wistar rats (APO-SUS) display a number of behavioral and pathophysiological features reminiscent of such disorders. Here, we report microarray analyses revealing in APO-SUS rats, relative to their counterpart APO-UNSUS rats, a reduced expression of Aph-1b, a component of the gamma-secretase enzyme complex that is involved in multiple (neuro)developmental signaling pathways. The reduced expression is due to a duplicon-based genomic rearrangement event resulting in an Aph-1b dosage imbalance. The expression levels of the other gamma-secretase components were not affected. However, gamma-secretase cleavage activity was significantly changed, and the APO-SUS/-UNSUS Aph-1b genotypes segregated with a number of behavioral phenotypes. Thus, a subtle imbalance in the expression of a single, developmentally important protein may be sufficient to cause a complex phenotype.

Tissue-specific inactivation of murine M6P/IGF2R.

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and T cell- mediated immunity. M6P/IGF2R is an imprinted gene in mice with expression only from the maternal allele. Complete knockout of this gene causes neonatal lethality, thus preventing analysis of its multifunctional role postnatally. To help elucidate the biological functions of M6P/IGF2R in adulthood, we generated both complete and tissue-specific M6P/IGF2R knockout mice using the Cre/loxP system. We confirm that complete M6P/IGF2R knockout results in fetal overgrowth and neonatal lethality. In contrast, tissue-specific inactivation of this gene in either the liver or skeletal and cardiac muscle gives rise to viable animals with no obvious phenotype. The successful creation of viable tissue-specific M6P/IGF2R knockout mouse models will now allow for detailed analysis of receptor function in a number of cellular processes including brain development, carcinogenesis, lysosomal trafficking, and T cell-mediated immunity.