Investigation of Parameters that Affect the Success Rate of Microarray-Based Allele-Specific Hybridization Assays

BACKGROUND: The development of microarray-based genetic tests for diseases that are caused by known mutations is becoming increasingly important. The key obstacle to developing functional genotyping assays is that such mutations need to be genotyped regardless of their location in genomic regions. These regions include large variations in G+C content, and structural features like hairpins. METHODS/FINDINGS: We describe a rational, stable method for screening and combining assay conditions for the genetic analysis of 42 Phenylketonuria-associated mutations in the phenylalanine hydroxylase gene. The mutations are located in regions with large variations in G+C content (20-75%). Custom-made microarrays with different lengths of complementary probe sequences and spacers were hybridized with pooled PCR products of 12 exons from each of 38 individual patient DNA samples. The arrays were washed with eight buffers with different stringencies in a custom-made microfluidic system. The data were used to assess which parameters play significant roles in assay development. CONCLUSIONS: Several assay development methods found suitable probes and assay conditions for a functional test for all investigated mutation sites. Probe length, probe spacer length, and assay stringency sufficed as variable parameters in the search for a functional multiplex assay. We discuss the optimal assay development methods for several different scenarios

Headache, tinnitus and hearing loss in the international Cohort Study of Mobile Phone Use and Health (COSMOS) in Sweden and Finland

Background Mobile phone use and exposure to radiofrequency electromagnetic fields (RF-EMF) from it have been associated with symptoms in some studies, but the studies have shortcomings and their findings are inconsistent. We conducted a prospective cohort study to assess the association between amount of mobile phone use at baseline and frequency of headache, tinnitus or hearing loss at 4-year follow-up. Methods The participants had mobile phone subscriptions with major mobile phone network operators in Sweden (n = 21 049) and Finland (n = 3120), gave consent for obtaining their mobile phone call data from operator records at baseline, and filled in both baseline and follow-up questionnaires on symptoms, potential confounders and further characteristics of their mobile phone use. Results The participants with the highest decile of recorded call-time (average call-time >276 min per week) at baseline showed a weak, suggestive increased frequency of weekly headaches at 4-year follow-up (adjusted odds ratio 1.13, 95% confidence interval 0.95–1.34). There was no obvious gradient of weekly headache with increasing call-time (P trend 0.06). The association of headache with call-time was stronger for the Universal Mobile Telecommunications System (UMTS) network than older Global System for Mobile Telecommunications (GSM) technology, despite the latter involving higher exposure to RF-EMF. Tinnitus and hearing loss showed no association with call-time. Conclusions People using mobile phones most extensively for making or receiving calls at baseline reported weekly headaches slightly more frequently at follow-up than other users, but this finding largely disappeared after adjustment for confounders and was not related to call-time in GSM with higher RF-EMF exposure. Tinnitus and hearing loss were not associated with amount of call-time

Microfludic Device for Creating Ionic Strength Gradients over DNA Microarrays for Efficient DNA Melting Studies and Assay Development

The development of DNA microarray assays is hampered by two important aspects: processing of the microarrays is done under a single stringency condition, and characteristics such as melting temperature are difficult to predict for immobilized probes. A technical solution to these limitations is to use a thermal gradient and information from melting curves, for instance to score genotypes. However, application of temperature gradients normally requires complicated equipment, and the size of the arrays that can be investigated is restricted due to heat dissipation. Here we present a simple microfluidic device that creates a gradient comprising zones of defined ionic strength over a glass slide, in which each zone corresponds to a subarray. Using this device, we demonstrated that ionic strength gradients function in a similar fashion as corresponding thermal gradients in assay development. More specifically, we noted that (i) the two stringency modulators generated melting curves that could be compared, (ii) both led to increased assay robustness, and (iii) both were associated with difficulties in genotyping the same mutation. These findings demonstrate that ionic strength stringency buffers can be used instead of thermal gradients. Given the flexibility of design of ionic gradients, these can be created over all types of arrays, and encompass an attractive alternative to temperature gradients, avoiding curtailment of the size or spacing of subarrays on slides associated with temperature gradients

Multi-stringency wash of partially hybridized 60-mer probes reveals that the stringency along the probe decreases with distance from the microarray surface

Here, we describe a multi-parametric study of DNA hybridization to probes with 20–70% G + C content. Probes were designed towards 71 different sites/mutations in the phenylalanine hydroxylase gene. Seven probe lengths, three spacer lengths and six stringencies were systematically varied. The three spacer lengths were obtained by placing the gene-specific sequence in discrete steps along the 60-mer probes. The study was performed using Agilent 8 × 15 000 probes custom-made arrays and a home-built array washer providing different stringencies to each of the eight sub-arrays on the slides. Investigation of hybridization signals, specificity and dissociation curves indicated that probes close to the surface were influenced by an additional stringency provided by the microarray surface. Consistent with this, probes close to the surface required 4 × SSC, while probes placed away from the surface required 0.35 × SSC wash buffers in order to give accurate genotyping results. Multiple step dissociation was frequently observed for probes placed furthest away from surface, but not for probes placed proximal to the surface, which is consistent with the hypothesis that there is different stringency along the 60-mer. The results have impact on design of probes for genotyping, gene expression and comparative genome hybridization analysis

Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders

Genetic influences on psychiatric disorders transcend diagnostic boundaries, suggesting substantial pleiotropy of contributing loci. However, the nature and mechanisms of these pleiotropic effects remain unclear. We performed analyses of 232,964 cases and 494,162 controls from genome-wide studies of anorexia nervosa, attention-deficit/hyper-activity disorder, autism spectrum disorder, bipolar disorder, major depression, obsessive-compulsive disorder, schizophrenia, and Tourette syndrome. Genetic correlation analyses revealed a meaningful structure within the eight disorders, identifying three groups of inter-related disorders. Meta-analysis across these eight disorders detected 109 loci associated with at least two psychiatric disorders, including 23 loci with pleiotropic effects on four or more disorders and 11 loci with antagonistic effects on multiple disorders. The pleiotropic loci are located within genes that show heightened expression in the brain throughout the lifespan, beginning prenatally in the second trimester, and play prominent roles in neurodevelopmental processes. These findings have important implications for psychiatric nosology, drug development, and risk prediction.Peer reviewe

IT-utveckling, systemutveckling för alla - även handikappade

Informationsteknologin breder ut sig på alla områden, men oftast är den utformad på ett sådant sätt, att vissa handikappade inte kan använda den. I vår uppsats uppmärksammas problem hos två grupper av handikappade: dyslektiker och synskadade. Dessa grupper kan behöva en särskild utformning av IT-systemen. Krav på att fokusera mer på tillgänglighet, användbarhet och användarcentrering har ökat från många olika håll. Lagar och Internationella Standardiserings Organisationen (ISO), kräver nu att system som byggs måste vara tillgängliga och användbara för alla människor, även handikappade.Användarcentrerad systemdesign innebär ett synsätt, som fokuserar på användbarhet, användarmedverkan och användarcentrering. I en kommersiell systemutvecklingsprocess, som Rational Unified Process (RUP), har man inte explicit tagit hänsyn till handikappade. Vi ger förslag på hur RUP med hjälp av användarcentrerade metoder, ISO- standarder och lagar, skall kunna erbjuda möjligheter att utveckla tillgängligare system som kan användas även av handikappade användare