Homogeneous Assays for Simplified Screening of HLA-conferred Genetic Disease Risk

The increasing incidence of type 1 diabetes has led researchers on a quest to find the reason behind this phenomenon. The rate of increase is too great to be caused simply by changes in the genetic component, and many environmental factors are under investigation for their possible contribution. These studies require, however, the participation of those individuals most likely to develop the disease, and the approach chosen by many is to screen vast populations to find persons with increased genetic risk factors. The participating individuals are then followed for signs of disease development, and their exposure to suspected environmental factors is studied. The main purpose of this study was to find a suitable tool for easy and inexpensive screening of certain genetic risk markers for type 1 diabetes. The method should be applicable to using whole blood dried on sample collection cards as sample material, since the shipping and storage of samples in this format is preferred. However, the screening of vast sample libraries of extracted genomic DNA should also be possible, if such a need should arise, for example, when studying the effect of newly discovered genetic risk markers. The method developed in this study is based on homogeneous assay chemistry and an asymmetrical polymerase chain reaction (PCR). The generated singlestranded PCR product is probed by lanthanide-labelled, LNA (locked nucleic acid)-spiked, short oligonucleotides with exact complementary sequences. In the case of a perfect match, the probe is hybridised to the product. However, if even a single nucleotide difference occurs, the probe is bound instead of the PCR product to a complementary quencher-oligonucleotide labelled with a dabcyl-moiety, causing the signal of the lanthanide label to be quenched. The method was applied to the screening of the well-known type 1 diabetes risk alleles of the HLA-DQB1 gene. The method was shown to be suitable as an initial screening step including thousands of samples in the scheme used in the TEDDY (The Environmental Determinants of Diabetes in the Young) study to identify those individuals at increased genetic risk. The method was further developed into dry-reagent form to allow an even simpler approach to screening. The reagents needed in the assay were in dry format in the reaction vessel, and performing the assay required only the addition of the sample and, if necessary, water to rehydrate the reagents. This allows the assay to be successfully executed even by a person with minimal laboratory experience.Siirretty Doriast

Oulun kaupungin turvapalvelulaitteiden selvitystyö

Tämän opinnäytetyön aiheena oli tehdä selvitystyö Oulun kaupungin turvapalvelun turvapuhelinratkaisuista. Oulun kaupungin turvapalvelujen hankintakausi päättyi vuoden 2010 alussa ja perinteisen turvapuhelimen lisäksi tarjouspyynnössä oli mukana lisäpalveluna helppokäyttöinen GSM-puhelin, telecare-tyyppinen turvapuhelinratkaisu, GPS-henkilöpaikannin ohjelmistoineen, IP-turvapuhelin ja IP-kameravalvonta tilaajan määräämistä tahoista. Lisäpalveluna olevia turvapuhelinratkaisuja käytetään turvapuhelinasiakkailla yksilöllisen tarpeen mukaan. Käyttäjänä voi olla myös turvapalvelujen auttajataho tai kotihoidon henkilöstö. Palvelutarpeen arvioinnissa on asiakkaan ideaalisimman turvapuhelinratkaisun määrittelemiseksi oltava tietoinen käytössä olevien laitteiden ominaisuuksista. Opinnäytetyö aloitettiin tutustumalla Oulun kaupungin turvapalvelun toimintaan ja turvapalvelujen hankinnan tarjousasiakirjoihin. Tämän jälkeen tutustuttiin turvapuhelinratkaisuihin, jotka turvapalvelulla oli käytettävissä. Opinnäytetyön tuloksena pidettiin kolme turvalaitteiden esittelytilaisuutta. Tilaisuudet oli tarkoitettu henkilöille, joiden tuli olla tietoisia turvapalveluratkaisuista, eli esimerkiksi palvelutarpeenarvioijille. Lisäksi turvalaitteista laadittiin kirjallinen materiaali, jota voidaan hyödyntää turvalaitteiden esittelyssä

Associations between deduced first islet specific autoantibody with sex, age at diagnosis and genetic risk factors in young children with type 1 diabetes

Objectives We aimed to further characterize demography and genetic associations of type 1 diabetes "endotypes" defined by the first appearing islet specific autoantibodies. Research Design and Methods We analyzed 3277 children diagnosed before the age of 10 years from the Finnish Pediatric Diabetes Register. The most likely first autoantibody could be deduced in 1636 cases (49.9%) based on autoantibody combinations at diagnosis. Distribution of age, sex, HLA genotypes and allele frequencies of 18 single nucleotide polymorphisms (SNPs) in non-HLA risk genes were compared between the endotypes. Results Two major groups with either glutamic acid decarboxylase (GADA) or insulin autoantibodies (IAA) as the deduced first autoantibody showed significant differences in their demographic and genetic features. Boys and children diagnosed at young age had more often IAA-initiated autoimmunity whereas GADA-initiated autoimmunity was observed more frequently in girls and in subjects diagnosed at an older age. IAA as the first autoantibody was also most common in HLA genotype groups conferring high-disease risk while GADA first was seen more evenly and frequently in HLA groups associated with lower type 1 diabetes risk. The risk alleles in IKZF4 and ERBB3 genes were associated with GADA-initiated whereas those in PTPN22, INS and PTPN2 genes were associated with IAA-initiated autoimmunity. Conclusions The results support the assumption that in around half of the young children the first autoantibody can be deduced based on islet autoantibody combinations at disease diagnosis. Strong differences in sex and age distributions as well as in genetic associations could be observed between GADA- and IAA-initiated autoimmunity.Peer reviewe

Associations between deduced first islet specific autoantibody with sex, age at diagnosis and genetic risk factors in young children with type 1 diabetes

Objectives We aimed to further characterize demography and genetic associations of type 1 diabetes "endotypes" defined by the first appearing islet specific autoantibodies. Research Design and Methods We analyzed 3277 children diagnosed before the age of 10 years from the Finnish Pediatric Diabetes Register. The most likely first autoantibody could be deduced in 1636 cases (49.9%) based on autoantibody combinations at diagnosis. Distribution of age, sex, HLA genotypes and allele frequencies of 18 single nucleotide polymorphisms (SNPs) in non-HLA risk genes were compared between the endotypes. Results Two major groups with either glutamic acid decarboxylase (GADA) or insulin autoantibodies (IAA) as the deduced first autoantibody showed significant differences in their demographic and genetic features. Boys and children diagnosed at young age had more often IAA-initiated autoimmunity whereas GADA-initiated autoimmunity was observed more frequently in girls and in subjects diagnosed at an older age. IAA as the first autoantibody was also most common in HLA genotype groups conferring high-disease risk while GADA first was seen more evenly and frequently in HLA groups associated with lower type 1 diabetes risk. The risk alleles in IKZF4 and ERBB3 genes were associated with GADA-initiated whereas those in PTPN22, INS and PTPN2 genes were associated with IAA-initiated autoimmunity. Conclusions The results support the assumption that in around half of the young children the first autoantibody can be deduced based on islet autoantibody combinations at disease diagnosis. Strong differences in sex and age distributions as well as in genetic associations could be observed between GADA- and IAA-initiated autoimmunity.Peer reviewe

Age at Seroconversion, HLA Genotype, and Specificity of Autoantibodies in Progression of Islet Autoimmunity in Childhood

Context: Children with initial autoantibodies to either insulin (IAA) or glutamic acid decarboxylase (GADA) differ in peak age of seroconversion and have different type 1 diabetes (T1D) risk gene associations, suggesting heterogeneity in the disease process. Objective: To compare the associations of age at seroconversion, HLA risk, and specificity of secondary autoantibodies with the progression of islet autoimmunity between children with either IAA or GADA as their first autoantibody. Design and methods: A cohort of 15,253 children with HLA-associated increased risk of T1D participated in a follow-up program in which islet autoantibodies were regularly measured. The median follow-up time was 6.7 years. Spearman correlation, Kaplan-Meier survival plots, and Cox proportional-hazard models were used for statistical analyses. Results: Persistent positivity for at least one of the tested autoantibodies was detected in 998 children; 388 of children progressed to clinical T1D. Young age at initial seroconversion was associated with a high probability of expansion of IAA-initiated autoimmunity and progression to clinical diabetes, whereas expansion of GADA-initiated autoimmunity and progression to diabetes were not dependent on initial seroconversion age. The strength of HLA risk affected the progression of both IAA- and GADA-initiated autoimmunity. The simultaneous appearance of two other autoantibodies increased the rate of progression to diabetes compared with that of a single secondary autoantibody among subjects with GADA-initiated autoimmunity but not among those with IAA as the first autoantibody. Conclusions: Findings emphasize the differences in the course of islet autoimmunity initiated by either IAA or GADA supporting heterogeneity in the pathogenic process.Peer reviewe

Tri-SNP polymorphism in the intron of HLA-DRA1 affects type 1 diabetes susceptibility in the Finnish population

Genes in the HLA class II region include the most important inherited risk factors for type 1 diabetes (T1D) although also polymorphisms outside the HLA region modulate the predisposition to T1D. This study set out to confirm a recent observation in which a novel expression quantitative trait locus was formed by three single nucleotide polymorphisms (SNP) in the intron of HLA-DRA1 in DR3-DQ2 haplotypes. The SNPs significantly increased the risk for T1D in DR3-DQ2 homozygous individuals and we intended to further explore this association, in the Finnish population, by comparing two DR3-DQ2 positive genotypes. Cohorts with DR3-DQ2/DR3-DQ2 (N = 570) and DR3-DQ2/DR1-DQ5 (N = 1035) genotypes were studied using TaqMan analysis that typed for rs3135394, rs9268645 and rs3129877. The tri-SNP haplotype was significantly more common in cases than controls in the DR3-DQ2/DR3-DQ2 cohort (OR = 1.70 CI 95% = 1.15-2.51P = 0.007). However, no significant associations could be observed in the DR3-DQ2/DR1-DQ5 cohort. (c) 2021 The Authors. Published by Elsevier Inc. on behalf of American Society for Histocompatibility and Immunogenetics. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).Peer reviewe

Associations between deduced first islet specific autoantibody with sex, age at diagnosis and genetic risk factors in young children with type 1 diabetes

Objectives We aimed to further characterize demography and genetic associations of type 1 diabetes "endotypes" defined by the first appearing islet specific autoantibodies. Research Design and Methods We analyzed 3277 children diagnosed before the age of 10 years from the Finnish Pediatric Diabetes Register. The most likely first autoantibody could be deduced in 1636 cases (49.9%) based on autoantibody combinations at diagnosis. Distribution of age, sex, HLA genotypes and allele frequencies of 18 single nucleotide polymorphisms (SNPs) in non-HLA risk genes were compared between the endotypes. Results Two major groups with either glutamic acid decarboxylase (GADA) or insulin autoantibodies (IAA) as the deduced first autoantibody showed significant differences in their demographic and genetic features. Boys and children diagnosed at young age had more often IAA-initiated autoimmunity whereas GADA-initiated autoimmunity was observed more frequently in girls and in subjects diagnosed at an older age. IAA as the first autoantibody was also most common in HLA genotype groups conferring high-disease risk while GADA first was seen more evenly and frequently in HLA groups associated with lower type 1 diabetes risk. The risk alleles in IKZF4 and ERBB3 genes were associated with GADA-initiated whereas those in PTPN22, INS and PTPN2 genes were associated with IAA-initiated autoimmunity. Conclusions The results support the assumption that in around half of the young children the first autoantibody can be deduced based on islet autoantibody combinations at disease diagnosis. Strong differences in sex and age distributions as well as in genetic associations could be observed between GADA- and IAA-initiated autoimmunity.</p

A characteristic time sequence of epileptic activity in EEG during dynamic penicillin-induced focal epilepsy—A preliminary study

AbstractPenicillin-induced focal epilepsy is a well-known model in experimental epilepsy. However, the dynamic evolution of waveforms, DC-level changes, spectral content and coherence are rarely reported. Stimulated by earlier fMRI findings, we also seek for the early signs preceding spiking activity from frequency domain of EEG signal. In this study, EEG data is taken from previous EEG/fMRI series (six pigs, 20–24kg) of an experimental focal epilepsy model, which includes dynamic induction of epileptic activity with penicillin (6000IU) injection into the somatosensory cortex during deep isoflurane anaesthesia. No ictal discharges were recorded with this dose. Spike waveforms, DC-level, time–frequency content and coherence of EEG were analysed. Development of penicillin induced focal epileptic activity was not preceded with specific spectral changes. The beginning of interictal spiking was related to power increase in the frequencies below 6Hz or 20Hz, and continued to a widespread spectral increase. DC-level and coherence changes were clear in one animal. Morphological evolution of epileptic activity was a collection of the low-amplitude monophasic, bipolar, triple or double spike-wave forms, with an increase in amplitude, up to large monophasic spiking. In conclusion, in the time sequence of induced epileptic activity, immediate shifts in DC-level EEG are plausible, followed by the spike activity-related widespread increase in spectral content. Morphological evolution does not appear to follow a clear continuum; rather, intermingled and variable spike or multispike waveforms generally lead to stabilised activity of high-amplitude monophasic spikes

HLA-DR-DQhaplotypes and specificity of the initial autoantibody in islet specific autoimmunity

Objective We aimed to clarify the association of various HLA risk alleles with different types of autoantibodies initiating islet specific autoimmunity. Methods Follow-up cohorts from the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study and children diagnosed with type 1 diabetes (T1D) from the Finnish Pediatric Diabetes Register (FPDR) were analyzed for the presence of autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), IA-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A); and genotyped for HLA DR/DQ alleles. In the DIPP study, autoantibodies were regularly analyzed from birth up to 15 years of age. Results In the DIPP cohort, 621 children developed one single persistent autoantibody, GADA in 284, IAA in 268, and IA-2A in 40 cases. Highly significant differences in the specificity of the first autoantibody were observed between HLA genotypes. Homozygotes for the DR3-DQ2 haplotype had almost exclusively GADA as the first autoantibody, whereas a more even distribution between GADA and IAA was found in DR3-DQ2/DR4-DQ8 as well as DR3-DQ/x and DR4-DQ8/x genotypes (x referring to neutral haplotypes). In DR4-DQ8 positive genotypes with the DRB1*04:01 allele IAA was more often the first autoantibody than in DRB1*04:04 positive genotypes. Various neutral haplotypes also significantly affected the relative proportions of different initial autoantibodies. These findings were confirmed and expanded in a series of 1591 T1D children under the age of 10 years from FPDR. Conclusions These results emphasize the importance of HLA class II polymorphisms in the recognition of autoantigen epitopes in the initiation of various pathways of the autoimmune response.Peer reviewe

Longitudinal Pattern of First-Phase Insulin Response Is Associated With Genetic Variants Outside the Class II HLA Region in Children With Multiple Autoantibodies

A declining first-phase insulin response (FPIR) is associated with positivity for multiple islet autoantibodies, irrespective of class II HLA DR-DQ genotype. We examined the associations of FPIR with genetic variants outside the HLA DR-DQ region in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study in children with and without multiple autoantibodies. Association between FPIR and class I alleles A*24 and B*39 and eight single nucleotide polymorphisms outside the HLA region were analyzed in 438 children who had one or more FPIR results available after seroconversion. Hierarchical linear mixed models were used to analyze repeated measurements of FPIR. In children with multiple autoantibodies, the change in FPIR over time was significantly different between those with various PTPN2 (rs45450798), FUT2 (rs601338), CTSH (rs3825932), and IKZF4 (rs1701704) genotypes in at least one of the models. In general, children carrying susceptibility alleles for type 1 diabetes experienced a more rapid decline in insulin secretion compared with children without susceptibility alleles. The presence of the class I HLA A*24 allele was also associated with a steeper decline of FPIR over time in children with multiple autoantibodies. Certain genetic variants outside the class II HLA region may have a significant impact on the longitudinal pattern of FPIR.Peer reviewe