Validation, Deployment, and Real-World Implementation of a Modular Toolbox for Alzheimer's Disease Detection and Dementia Risk Reduction: The AD-RIDDLE Project.

The Real-World Implementation, Deployment, and Validation of Early Detection Tools and Lifestyle Enhancement (AD-RIDDLE) project, recently launched with the support of the EU Innovative Health Initiative (IHI) public-private partnership and UK Research and Innovation (UKRI), aims to develop, test, and deploy a modular toolbox platform that can reduce existing barriers to the timely detection, and therapeutic approaches in Alzheimer's disease (AD), thus accelerating AD innovation. By focusing on health system and health worker practices, AD-RIDDLE seeks to improve and smooth AD management at and between each key step of the clinical pathway and across the disease continuum, from at-risk asymptomatic stages to early symptomatic ones. This includes innovation and improvement in AD awareness, risk reduction and prevention, detection, diagnosis, and intervention. The 24 partners in the AD-RIDDLE interdisciplinary consortium will develop and test the AD-RIDDLE toolbox platform and its components individually and in combination in six European countries. Expected results from this cross-sectoral research collaboration include tools for earlier detection and accurate diagnosis; validated, novel digital cognitive and blood-based biomarkers; and improved access to individualized preventative interventions (including multimodal interventions and symptomatic/disease-modifying therapies) across diverse populations, within the framework of precision medicine. Overall, AD-RIDDLE toolbox platform will advance management of AD, improving outcomes for patients and their families, and reducing costs.

Recommendations for locus-specific databases and their curation.

Expert curation and complete collection of mutations in genes that affect human health is essential for proper genetic healthcare and research. Expert curation is given by the curators of gene-specific mutation databases or locus-specific databases (LSDBs). While there are over 700 such databases, they vary in their content, completeness, time available for curation, and the expertise of the curator. Curation and LSDBs have been discussed, written about, and protocols have been provided for over 10 years, but there have been no formal recommendations for the ideal form of these entities. This work initiates a discussion on this topic to assist future efforts in human genetics. Further discussion is welcome.

DFold: PCR design that minimizes secondary structure and optimizes downstream genotyping applications.

Secondary structures in polymerase chain reaction (PCR) target sequences have a negative impact on amplification success rates and on downstream uses of PCR products. For example, signal strength and allele discrimination in single nucleotide polymorphism (SNP) genotyping methods can be compromised by allele-biased amplification and/or by PCR product folding that limits access of interrogating probes. To increase the fidelity and robustness of PCR, and to aid follow-on applications, we have developed DFold (http://dfold.cgb.ki.se)-a generalized software solution that creates PCR oligonucleotide primer designs devoid of stable secondary structures. We demonstrate the effectiveness of the tool by applying it to a range of dynamic allele-specific hybridization (DASH) assay designs, many of which we evaluate in the laboratory. We further consider how the system throughput may be made sufficiently high for use upon millions of target sequences in order to support whole-genome analyses.

A novel screen for nuclear mitochondrial gene associations with Parkinson's disease.

Genetic factors play an important role in the aetiology of Parkinson's disease (PD). We have screened nuclear genes encoding subunits of mitochondrial complex I for associations between single nucleotide polymorphisms (SNPs) and PD. Abnormal functioning of complex I is well documented in human PD. Moreover, toxicological inhibition of complex I can lead to parkinsonism in animals. Thus, commonly occurring variants in these genes could potentially influence complex I function and the risk of developing PD. A sub-set of 70 potential SNPs in 31 nuclear complex I genes were selected and association analysis was performed on 306 PD patients plus 321 unaffected control subjects. Genotyping was performed using the DASH method. There was no evidence that the examined SNPs were significant genetic risk factors for PD, although this initial screen could not exclude the possibility that other disease-influencing variations exist within these genes.

HGVbase: a curated resource describing human DNA variation and phenotype relationships.

The Human Genome Variation Database (HGVbase; http://hgvbase.cgb.ki.se) has provided a curated summary of human DNA variation for more than 5 years, thus facilitating research into DNA sequence variation and human phenotypes. The database has undergone many changes and improvements to accommodate increasing volumes and new types of data. The focus of HGVbase has recently shifted towards information on haplotypes and phenotypes, relationships between phenotypes and DNA variation, and collaborative efforts to provide a global resource for genome-phenome data. Open sharing and precise phenotype definitions are necessary to advance the current understanding of common diseases that are typified by complex aetiologies, small genetic effect sizes and multiple confounding factors that obscure positive study results. Association data will increasingly be collected as part of this new project thrust. This report describes the evolving features of HGVbase, and covers in detail the technological choices we have made to enable efficient storage and data mining of increasingly large and complex data sets.

Evidence for an important role of perilipin in the regulation of human adipocyte lipolysis.

AIMS/HYPOTHESIS: We investigated the role of the adipocyte-specific protein perilipin for lipolysis in humans. METHODS: Perilipin protein content and lipolysis rates were measured in human subcutaneous fat cells of non-obese (n=10) and obese (n=117) women. Single nucleotide polymorphisms in the perilipin gene were examined in obese subjects. RESULTS: Basal and noradrenaline-induced rates of lipolysis were two to fourfold increased (p<0.01) and perilipin protein content decreased 50% (p=0.005) in adipocytes of the obese women. In subjects matched for body mass index and fat-cell volume, a high rate of lipolysis was associated with a low adipocyte content of perilipin (p=0.01). Adipocyte content of perilipin was inversely correlated with the circulating concentrations of glycerol (r=0.62) and non-esterified fatty acids (n=0.49). A gene polymorphism (rs891460 A/G) in intron 6 was common. In AA subjects basal and noradrenaline induced lipolysis were 50 to 100% times more rapid (p

HGVbase: a human sequence variation database emphasizing data quality and a broad spectrum of data sources.

HGVbase (Human Genome Variation database; http://hgvbase.cgb.ki.se, formerly known as HGBASE) is an academic effort to provide a high quality and non-redundant database of available genomic variation data of all types, mostly comprising single nucleotide polymorphisms (SNPs). Records include neutral polymorphisms as well as disease-related mutations. Online search tools facilitate data interrogation by sequence similarity and keyword queries, and searching by genome coordinates is now being implemented. Downloads are freely available in XML, Fasta, SRS, SQL and tagged-text file formats. Each entry is presented in the context of its surrounding sequence and many records are related to neighboring human genes and affected features therein. Population allele frequencies are included wherever available. Thorough semi-automated data checking ensures internal consistency and addresses common errors in the source information. To keep pace with recent growth in the field, we have developed tools for fully automated annotation. All variants have been uniquely mapped to the draft genome sequence and are referenced to positions in EMBL/GenBank files. Data utility is enhanced by provision of genotyping assays and functional predictions. Recent data structure extensions allow the capture of haplotype and genotype information, and a new initiative (along with BiSC and HUGO-MDI) aims to create a central repository for the broad collection of clinical mutations and associated disease phenotypes of interest.

Rethinking genetic strategies to study complex diseases.

Understanding the genetic basis of complex diseases is turning out to be difficult, prompting a widespread (re-)evaluation of the relevant issues. 'Forward' and 'reverse' genetics strategies have been applied arguably in a manner only suitable for much simpler diseases. It would now be beneficial to pay detailed attention to experimental design, and to increase study scales dramatically. Ultimately, this would lead to completely hypothesis-free, truly comprehensive, multi-platform investigations. Such studies would maximize the chances of finding data patterns indicative of real etiology, although many aspects of complex disease causation might simply be too intricate and inconsistent to ever be deciphered. Therefore, considerable technology development is an immediate priority, along with parallel advances in bioinformatics and biostatistics systems aimed at discriminating between marginal signals and background noise within extremely large, diverse and complex data sets. Community standards and open data sharing will be essential ingredients for success in this exciting 21st-century challenge.

Truncation of WT1 results in downregulation of cyclin G1 and IGFBP-4 expression.

Mutations in the WT1 gene are found in a subset of Wilms' tumours and in certain other disorders such as Denys-Drash syndrome. The WT1 gene product is a zinc finger transcription factor for which many target genes have been suggested. Here we utilise gene targeting to generate cells containing only truncated forms of WT1, in which the DNA-binding region is disrupted. Examination of gene expression in these cells using cDNA macroarrays suggests two novel WT1 transcriptional targets, cyclin G1 (Ccng1), and insulin-like growth factor binding protein 4 (Igfbp4).

Lack of replication of association findings in complex disease: an analysis of 15 polymorphisms in prior candidate genes for sporadic Alzheimer's disease.

There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

SNP association studies in Alzheimer's disease highlight problems for complex disease analysis.

Genetic linkage and association analyses are two distinct approaches to understanding the genetic etiology of complex disease. Association analysis has become particularly popular in recent times, but the true utility of the strategy remains uncertain. To try to gain better insight into the relevant issues, we have used genetic association analysis to explore the etiology of Alzheimer's disease. Our empirical findings supplement the theoretical debate, illustrating the general doubtfulness of previous positive findings and the limited ability of typical association studies based on candidate genes to discern true medium-sized signals from false positives. Improvements in genotyping technologies and increasing the number of SNPs tested, without sophisticated allowance for all other issues, could simply lead to an unmanageable overload of false-positive signals, themselves obscuring true disease associations.

3rd International Meeting on Single Nucleotide Polymorphism and Complex Genome Analysis: SNPs: 'some notable progress'.

Fervent activities for the collection and exploitation of single nucleotide polymorphism (SNP) data continue, amid concerns about their real utility. The desire to understand complex disease aetiology remains a key driving force for this activity. Recent developments provided a level of cautious optimism not seen in previous International Meetings on Single Nucleotide Polymorphism and Complex Genome Analysis. The 3rd such meeting, held 8-11 September 2000 in Taos, New Mexico, covered research on technologies for SNP scoring, analytical tools for using SNPs to map disease genes, examples from researchers using SNPs for specific disease studies, and databases and tools for facilitating these activities. Studies of human history, and a range of studies upon model organisms were also represented. Whilst the transition from technology oriented work (methods, discovery, etc.) to successful biological application is occurring relatively slowly, a clear trend in this direction is now apparent, and it will surely gain momentum in future months and years. Many fundamental properties of SNPs remain unknown, and many other basic questions are still unanswered, but the field is moving forward on all necessary fronts, promising exciting advances just around the corner.

The parkin gene S/N167 polymorphism in Australian Parkinson's disease patients and controls.

This study determined the frequencies of a G-to-A transition (S/N167) polymorphism in exon 4 of the parkin gene in Australian Parkinson's disease patients and control subjects. The genotype of each subject was determined using the polymerase chain reaction and restriction-fragment-length-polymorphism analysis. Overall, the A allele was significantly less common in the Parkinson's disease group (1.7%) compared with the control group (3.8%, OR=0.43, 95% CI=0.19-1.00, P<0.05), although the frequency in the young onset Parkinson's disease group (6.6%) was not significantly different to controls. The A allele is less common in Australian Caucasian subjects compared to Japanese Parkinson's disease patients and appears to be under-represented in older-onset Parkinson's disease.

Robust and accurate single nucleotide polymorphism genotyping by dynamic allele-specific hybridization (DASH): design criteria and assay validation.

We recently introduced a generic single nucleotide polymorphism (SNP) genotyping method, termed DASH (dynamic allele-specific hybridization), which entails dynamic tracking of probe (oligonucleotide) to target (PCR product) hybridization as reaction temperature is steadily increased. The reliability of DASH and optimal design rules have not been previously reported. We have now evaluated crudely designed DASH assays (sequences unmodified from genomic DNA) for 89 randomly selected and confirmed SNPs. Accurate genotype assignment was achieved for 89% of these worst-case-scenario assays. Failures were determined to be caused by secondary structures in the target molecule, which could be reliably predicted from thermodynamic theory. Improved design rules were thereby established, and these were tested by redesigning six of the failed DASH assays. This involved reengineering PCR primers to eliminate amplified target sequence secondary structures. This sophisticated design strategy led to complete functional recovery of all six assays, implying that SNPs in most if not all sequence contexts can be effectively scored by DASH. Subsequent empirical support for this inference has been evidenced by approximately 30 failure-free DASH assay designs implemented across a range of ongoing genotyping programs. Structured follow-on studies employed standardized assay conditions, and revealed that assay reproducibility (733 duplicated genotypes, six different assays) was as high as 100%, with an assay accuracy (1200 genotypes, three different assays) that exceeded 99.9%. No post-PCR assay failures were encountered. These findings, along with intrinsic low cost and high flexibility, validate DASH as an effective procedure for SNP genotyping.

Towards high-throughput genotyping of SNPs by dynamic allele-specific hybridization.

Analysis of single nucleotide polymorphisms (SNPs)--the most common form of variation in the human genome--has become a popular strategy for discovering genes involved in complex diseases such as Alzheimer's disease, obesity and diabetes. It is also widely anticipated that SNPs will play a major role in pharmacogenomics, where the identification of variations in specific genes relevant to drug efficacy, toxicity and metabolism will help to establish optimal therapeutic strategies for individual patients. Reflecting these expectations, many new SNP-related technologies have appeared over the past few years, each with unique advantages, but all with the common goal of simplifying and expediting SNP analysis. We recently introduced a technique termed dynamic allele-specific hybridization (DASH), a convenient method for SNP (and insertion-deletion) genotyping, which is highly applicable to both basic research and clinical diagnostics. Commercial DASH devices are now available, making the technology affordably accessible for all laboratories.

apolipoprotein-E dependent role for the FAS receptor in early onset Alzheimer's disease: finding of a positive association for a polymorphism in the TNFRSF6 gene.

The TNFRSF6 gene encodes FAS, a cell-surface receptor involved in apoptosis initiation. Elevated levels of FAS have been reported in the brains of Alzheimer's disease (AD) patients. We have tested a G/A polymorphism at position -670 in the TNFRSF6 gene for association with non-familial, early onset Alzheimer's disease (EOAD) by using dynamic allele-specific hybridization. In an initial set of Scottish EOAD cases (n=78) and controls (n=152), we found that, for individuals carrying one or two APOE4 alleles, the homozygous GG-genotype was enriched in the patients (26.7% versus 10.9% in controls). A second study was conducted on an independent set of Scottish individuals (87 EOAD, 358 controls). In this material, the TNFRSF6 GG-genotype frequency was elevated in patients regardless of APOE4 status (28.7% versus 15.1%) and was even more enriched in APOE4 carriers (35.9% versus 15.3%). A combination of the two sample sets (165 cases, 510 controls) gave a significant disease association for the TNFRSF6 GG-genotype that was irrespective of APOE4 (P=0.0020) and that was almost completely attributable to the enrichment present within the set of APOE4 carriers (P=0.0016). This represents an odds ratio of 8.71 for GG-homozygotes carrying at least one APOE4 allele compared with other TNFRSF6 genotypes in APOE4 non-carriers. The TNFRSF6 variation was further explored in Scottish late-onset Alzheimer's disease (n=159) but no associations were found. These results imply that TNFRSF6, in interaction with APOE4, is a genetic risk factor for sporadic EOAD. Hence, the AD risk contributed by APOE4 could be mechanistically related to a pathway in common with FAS-mediated apoptosis.