Polymorphisms in the Innate Immune IFIH1 Gene, Frequency of Enterovirus in Monthly Fecal Samples during Infancy, and Islet Autoimmunity

Interferon induced with helicase C domain 1 (IFIH1) senses and initiates antiviral activity against enteroviruses. Genetic variants of IFIH1, one common and four rare SNPs have been associated with lower risk for type 1 diabetes. Our aim was to test whether these type 1 diabetes-associated IFIH1 polymorphisms are associated with the occurrence of enterovirus infection in the gut of healthy children, or influence the lack of association between gut enterovirus infection and islet autoimmunity

Human blood RNA stabilization in samples collected and transported for a large biobank

Background: The Norwegian Mother and Child Cohort Study (MoBa) is a nation-wide population-based pregnancy cohort initiated in 1999, comprising more than 108.000 pregnancies recruited between 1999 and 2008. In this study we evaluated the feasibility of integrating RNA analyses into existing MoBa protocols. We compared two different blood RNA collection tube systems – the PAXgene™ Blood RNA system and the Tempus™ Blood RNA system - and assessed the effects of suboptimal blood volumes in collection tubes and of transportation of blood samples by standard mail. Endpoints to characterize the samples were RNA quality and yield, and the RNA transcript stability of selected genes. Findings: High-quality RNA could be extracted from blood samples stabilized with both PAXgene and Tempus tubes. The RNA yields obtained from the blood samples collected in Tempus tubes were consistently higher than from PAXgene tubes. Higher RNA yields were obtained from cord blood (3 – 4 times) compared to adult blood with both types of tubes. Transportation of samples by standard mail had moderate effects on RNA quality and RNA transcript stability; the overall RNA quality of the transported samples was high. Some unexplained changes in gene expression were noted, which seemed to correlate with suboptimal blood volumes collected in the tubes. Temperature variations during transportation may also be of some importance. Conclusions: Our results strongly suggest that special collection tubes are necessary for RNA stabilization and they should be used for establishing new biobanks. We also show that the 50,000 samples collected in the MoBa biobank provide RNA of high quality and in sufficient amounts to allow gene expression analyses for studying the association of disease with altered patterns of gene expression

Long-term storage of blood RNA collected in RNA stabilizing Tempus tubes in a large biobank – evaluation of RNA quality and stability

Background: Establishing methods for secure long term storage of RNA is critical to realizing the promise of biobanks in biomedical research. Here, we describe the results of yearly analyses of the same set of umbilical cord and adult whole blood RNA collected in Tempus Blood RNA tubes and stored at -80°C, over a period of up to six years. We systematically investigated the effects of long-term storage of samples (75 Tempus tubes form three adult donors and 30 Tempus tubes from three cord blood donors) on the RNA quality and transcript stability of six selected genes (CDKN1A, FOS, IL1B, IL8, MYC and TP53). This is the first systematic study of both cord and adult blood samples stored for many years. Findings: The RNA purity and integrity, expressed as RIN-values, were stable up to six years of storage, and there were no storage-related deleterious effects on RNA purity. There were limited intra- and inter-individual variations in RNA yields; however, no consistent trend of decreasing RNA yield was observed with the duration of storage. Some long-term storage effects were found on the relative transcript levels of the six genes when compared to the year 0 samples. However, these changes were within ± 2–fold for both types of blood samples, except for two genes. Our results show that storage of these samples for up to six years did not have significant effects on the RNA quality and transcript stability of the six genes. Conclusions: Blood RNA is stable in Tempus tubes stored at -80°C over a period of six years. Intact and good-quality RNA suitable for transcript profiling analyses in epidemiological studies was obtained from blood samples stored in Tempus tubes. This suggests that blood samples collected in large biobanks–such as the Mother and Child (MoBa) Cohort at Norwegian Institute of Public Health (NIPH) and frozen in suitable collection tubes for total RNA stabilization, can be used for quantitative studies after at least six years of storage

Construction and analysis of tag single nucleotide polymorphism maps for six human-mouse orthologous candidate genes in type 1 diabetes.

BACKGROUND: One strategy to help identify susceptibility genes for complex, multifactorial diseases is to map disease loci in a representative animal model of the disorder. The nonobese diabetic (NOD) mouse is a model for human type 1 diabetes. Linkage and congenic strain analyses have identified several NOD mouse Idd (insulin dependent diabetes) loci, which have been mapped to small chromosome intervals, for which the orthologous regions in the human genome can be identified. Here, we have conducted re-sequencing and association analysis of six orthologous genes identified in NOD Idd loci: NRAMP1/SLC11A1 (orthologous to Nramp1/Slc11a1 in Idd5.2), FRAP1 (orthologous to Frap1 in Idd9.2), 4-1BB/CD137/TNFRSF9 (orthologous to 4-1bb/Cd137/Tnrfrsf9 in Idd9.3), CD101/IGSF2 (orthologous to Cd101/Igsf2 in Idd10), B2M (orthologous to B2m in Idd13) and VAV3 (orthologous to Vav3 in Idd18). RESULTS: Re-sequencing of a total of 110 kb of DNA from 32 or 96 type 1 diabetes cases yielded 220 single nucleotide polymorphisms (SNPs). Sixty-five SNPs, including 54 informative tag SNPs, and a microsatellite were selected and genotyped in up to 1,632 type 1 diabetes families and 1,709 cases and 1,829 controls. CONCLUSION: None of the candidate regions showed evidence of association with type 1 diabetes (P values > 0.2), indicating that common variation in these key candidate genes does not play a major role in type 1 diabetes susceptibility in the European ancestry populations studied.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

HTR1A a Novel Type 1 Diabetes Susceptibility Gene on Chromosome 5p13-q13

Background: We have previously performed a genome-wide linkage study in Scandinavian Type 1 diabetes (T1D) families. In the Swedish families, we detected suggestive linkage (LOD less than= 2.2) to the chromosome 5p13-q13 region. The aim of our study was to investigate the linked region in search for possible T1D susceptibility genes. Methodology/Principal Findings: Microsatellites were genotyped in the Scandinavian families to fine-map the previously linked region. Further, SNPs were genotyped in Swedish and Danish families as well as Swedish sporadic cases. In the Swedish families we detected genome-wide significant linkage to the 5-hydroxytryptamine receptor 1A (HTR1A) gene (LOD 3.98, pless than9.8x10(-6)). Markers tagging two separate genes; the ring finger protein 180 (RNF180) and HTR1A showed association to T1D in the Swedish and Danish families (pless than0.002, pless than0.001 respectively). The association was not confirmed in sporadic cases. Conditional analysis indicates that the primary association was to HTR1A. Quantitative PCR show that transcripts of both HTR1A and RNF180 are present in human islets of Langerhans. Moreover, immunohistochemical analysis confirmed the presence of the 5-HTR1A protein in isolated human islets of Langerhans as well as in sections of human pancreas. Conclusions: We have identified and confirmed the association of both HTR1A and RFN180, two genes in high linkage disequilibrium (LD) to T1D in two separate family materials. As both HTR1A and RFN180 were expressed at the mRNA level and HTR1A as protein in human islets of Langerhans, we suggest that HTR1A may affect T1D susceptibility by modulating the initial autoimmune attack or either islet regeneration, insulin release, or both