Direct Detection of Disease-Associated Prions in Brain and Lymphoid Tissue Using Antibodies Recognizing the Extreme N-terminus of PrPC

A simple diagnostic test is described for the detection of TSE in bovine, ovine and human brain and lymphoid tissue that obviates the use of proteinase K as a discriminating reagent. The immunoassay utilises high affinity anti-peptide antibodies that appear blind to the normal isoform of prion protein (PrP^C). These reagents have been produced with novel N-terminal chimeric peptides and we hypothesise that the retention and stability of the extreme N-terminus of PrP in the disease-associated aggregate makes it an operationally specific marker for TSE. Accordingly, the assay involves homogenisation of the tissue directly in 8M guanidine hydrochloride, a simple one-step capture of PrP^Sc followed by detection with a europium-labelled anti-PrP^C antibody. This rapid assay clearly differentiates between levels of disease-associated PrP extracted from brain and lymphoid tissues taken from confirmed TSE positive and negative cattle and sheep. The assay can also be used to detect PrP^Sc in cases of vCJD.     

Monoclonal Antibodies Recognizing the Benzodiazepine Receptor of Chick Embryo Brain

Abstract

The benzodiazepine receptor (BZDR) of the embryonic chick brain contained three subunit proteins with molecular weights of 48-kilodalton (KD), 50-KD and 51-KD at a pI of 5.6, as demonstrated by two-dimensional gel electrophoresis and fluorography of the ³H-flunitrazepam (FNZ)-photolabeled receptor. Monoclonal antibodies (mAB) against the receptor were produced by using the spleen cells of one mouse immunized with the three subunit proteins extracted from SDS-PAGE gels. When the radioligand-labeled membranes were subjected to two-dimensional gel electrophoresis followed by immunoblotting using the mAB 2C3, both 50-KD and 51-KD bands with a pI of 5.6 were immunoreactive and radioactive. Thus, the mAB 2C3 recognized a common epitope on the 50-KD and 51-KD subunits of the BZDR. In addition, the mAB 2C3 was used with immunocytochemistry to determine the distribution of the receptor in the chick embryo brain. The BZDR immunoreactivity was observed among various brain areas, including hippocampus, optic tectum and cerebellum. The reaction product was localized in the neuronal membranes and cytoplasm. Certain neurons in the culture derived from embryonic chick brains were also immunoreactive as detected by immunocytochemical staining.     

Antibodies to the Buried N Terminus of Rhinovirus VP4 Exhibit Cross-Serotypic Neutralization

Development of a vaccine for the common cold has been thwarted by the fact that there are more than 100 serotypes of human rhinovirus (HRV). We previously demonstrated that the HRV14 capsid is dynamic and transiently displays the buried N termini of viral protein 1 (VP1) and VP4. Here, further evidence for this “breathing” phenomenon is presented, using antibodies to several peptides representing the N terminus of VP4. The antibodies form stable complexes with intact HRV14 virions and neutralize infectivity. Since this region of VP4 is highly conserved among all of the rhinoviruses, antiviral activity by these anti-VP4 antibodies is cross-serotypic. The antibodies inhibit HRV16 infectivity in a temperature- and time-dependent manner consistent with the breathing behavior. Monoclonal and polyclonal antibodies raised against the 30-residue peptide do not react with peptides shorter than 24 residues, suggesting that these peptides are adopting three-dimensional conformations that are highly dependent upon the length of the peptide. Furthermore, there is evidence that the N termini of VP4 are interacting with each other upon extrusion from the capsid. A Ser5Cys mutation in VP4 yields an infectious virus that forms cysteine cross-links in VP4 when the virus is incubated at room temperature but not at 4°C. The fact that all of the VP4s are involved in this cross-linking process strongly suggests that VP4 forms specific oligomers upon extrusion. Together these results suggest that it may be possible to develop a pan-serotypic peptide vaccine to HRV, but its design will likely require details about the oligomeric structure of the exposed termini.Rhinoviruses are the major causative agents of the common cold and cost the United States economy approximately $40 billion per year (6). Therefore, it is of great interest to prevent or ameliorate the symptoms of the common cold. The rhinovirus genus is a member of the picornavirus family and is characterized by nonenveloped capsid with a diameter of ∼300 Å containing a single-stranded, plus-sense RNA genome (19). Other members of the picornavirus family include foot-and-mouth disease virus, poliovirus, encephalomyocarditis virus, and hepatitis A virus. The capsids exhibit pseudo T = 3 icosahedral symmetry and are composed of 60 copies of the four capsid proteins VP1, VP2, VP3, and VP4. VP1, VP2, and VP3 have an eight-stranded antiparallel beta-barrel motif structure and form the outer surface of the capsid, while VP4 lies at the interface between the capsid and the interior genomic RNA (22). VP4 is approximately 70 amino acids in length and is myristoylated at the N terminus (3, 14).Antibodies are the major line of defense against picornavirus infections. In the case of human rhinovirus 14 (HRV14), a number of studies have been performed to detail the antibody recognition and neutralization processes (25). While it had been long suggested that antibodies neutralize viral infectivity by inducing large conformational changes in the capsid, both cryo-transmission electron microscopy (cryo-TEM) (2, 28) and crystallographic analysis (27) clearly demonstrated that this was not the case. Further, it was shown that antibody recognition is more plastic than previously thought in that it is able to bind into the relatively narrow receptor-binding region of the canyon (27). These results suggested that the major in vivo role of antibodies is to bind to virion and work synergistically with other immune system components (26). This hypothesis has gained further support from studies of other pathogens (1) and implies that vaccines need only to elicit antibodies that bind to the authentic pathogen with high affinity.While these results simplified the goal of creating a synthetic vaccine by focusing on capsid recognition rather than possible antibody-induced conformational changes, developing synthetic vaccines against all 100 serotypes of HRV remains a daunting task. As shown in the structures of HRV14/antibody complexes, the antibodies make extensive contacts with the surface of the capsid that is not limited to a single antigenic loop (2, 27). Further evidence for this extensive contact is that antibodies to peptides corresponding to antigenic NIm loops fail to neutralize the virions (17, 29), and antibodies raised against intact capsids do not bind effectively to peptides corresponding to NIm-IA loop (T. J. Smith, unpublished results). One notable exception is the case of HRV2, where there is cross-reactivity between the NIm-II site of the virion and a synthetic peptide (30). Nevertheless, developing a repertoire of peptides representing the entire antigenic ensemble of HRVs is not only impractical but also unlikely to elicit neutralizing antibodies.All of the studies described above were performed with the antibodies that were raised against intact particles or to peptides representing epitopes that reside on the outer surface of the capsid. In the case of poliovirus, however, antibodies were raised against VP4 and the N termini of VP1 of poliovirus serotype I (15, 21). It was shown that these antibodies are capable of neutralizing the virion despite the fact that those portions of the capsid protein are buried in the interior of the capsid at the capsid-RNA interface (8). These results suggested that the poliovirus capsid was more dynamic than indicated by the crystal structure and that these termini are presented to the exterior of the virion in a temperature-dependent and reversible manner. While the role of capsid dynamics in the viral life cycle was not clear, it was suggested that the N termini of VP1 and VP4 might facilitate cell membrane attachment and subsequent entry of the virus into the host cell (3, 4).More recently, evidence for capsid dynamics has been found in other viruses as well. In the cases of swine vesicular disease virus (10) and coxsackievirus A9 (18), antibodies were raised against the whole virus in pigs and rabbits, respectively. These polyclonal antibodies demonstrated a strong reaction to the peptides corresponding to the N termini of VP1 and VP3 of swine vesicular disease virus and coxsackievirus A9, respectively. In a similar study, antibodies from the plasma of patients suffering from type I diabetes were found to target VP4 protein of coxsackievirus B3, again suggesting the exposure of VP4 peptide during coxsackievirus infection (23). These results imply that capsid “breathing” may be a phenomenon common to many proteinaceous capsids.Using a very different approach, the dynamic nature of HRV14 was analyzed using limited proteolysis and mass spectrometry (matrix-assisted laser desorption ionization [MALDI]) analyses (14). In these experiments, the virus was treated with both matrix-bound and soluble forms of trypsin for various periods of time, and the resulting proteolytic fragments were identified by MALDI. Surprisingly, the N termini of VP4 and VP1 were found to be the most proteolytically sensitive portions of the capsid in spite of being buried inside the viral capsid. As an additional control, the antiviral “WIN” compounds, which had been previously shown to stabilize the virions against thermal and acid denaturation, were added during digestion. While these WIN compounds did not affect the intrinsic proteolytic activity of trypsin, they nearly completely protected the VP1 and VP4 termini from proteolysis for an extended period. Together, these results suggested that HRV14 is transiently exposing these termini in a “breathing” process and that the empty hydrophobic drug-binding region apparently plays an important role in facilitating these dynamics.In this study we further examined HRV14 capsid dynamics by raising polyclonal antibodies against several peptides representing the N termini of VP1 and VP4. In these experiments, only the antibodies against the VP4 N terminus were found to successfully neutralize viral infectivity in vitro. Further, we demonstrate that the HRV14 VP4 antiserum cross-reacts with other serotypes of rhinovirus (HRV16, and HRV29), which is likely due to the high degree of conservation of VP4. Antibody neutralization closely parallels the MALDI analysis in that antibody neutralization and proteolysis are enhanced at 37°C in the case of HRV16 whereas the elevated temperatures are not required for either phenomenon in the cases of HRV14 and HRV29. Epitope mapping of the N-terminal 30 residues of VP4 suggests that it adopts a nonlinear conformation, and this is further substantiated by results showing that all of the copies of VP4 in the Ser5Cys HRV14 mutant at room temperature form cysteine cross-linked dimers. This cysteine cross-link does not form at 4°C, suggesting that capsid breathing is essential for VP4 exposure and interactions. Since VP4 dimerization does not affect viral infectivity, it seems likely that VP4 extrusion is a normal part of the cell attachment and entry process of rhinovirus. Together, these results suggest that VP4 might be useful as a pan-serotypic rhinovirus vaccine, but it seems likely that better understanding of the VP4 oligomeric structure will be necessary for further optimization.     

Development and Characterization of Antibodies Against the N Terminus of the Human Dopamine D4 Receptor

Abstract: The human dopamine D4 receptor (hD4R), which has been implicated in human diseases such as schizophrenia and in a personality trait called "novelty seeking," has not yet been characterized at the protein level. Following epitope scanning of the hD4R, we have produced a highly specific monoclonal antibody named DFR1 raised against an amino-terminal peptide in a predicted extracellular region of the receptor. DFR1 decorated recombinant hD4Rs on the surface of intact Chinese hamster ovary (CHO) cells by flow cytometry and fluorescence microscopy and also recognized recombinant hD4.2, hD4.4, and hD4.7 receptor isoforms by western blot analysis. When expressed stably in CHO cells, all three hD4R isoforms contained N-linked glycosylation and showed apparent molecular masses of 48, 55, and 67 kDa for hD4.2, hD4.4, and hD4.7, respectively. DFR1 immunoreactivity representing hD4R protein or dopamine D4 receptor-like antigens was observed in crude membrane extracts of postmortem human brain tissue by immunoblotting. The DFR1 antibody provides a new immunological tool with the potential to further our understanding of the human dopamine D4 receptor protein.     

Clathrin-Coated Vesicle Subtypes in Mammalian Brain Tissue: Detection of Polypeptide Heterogeneity by Immunoprecipitation with Monoclonal Antibodies

A panel of monoclonal antibodies (mAbs) was developed to identify polypeptides sorted in subtypes of brain coated vesicles (CVs) and to separate these by immunoprecipitation. The corresponding antigen of some of the mAbs elicited by CV components was present also in synaptosomal plasma membrane, synaptic vesicles, or microsomes. On immunoblots the mAbs reacted with constitutive brain CV proteins, with cargo molecules, and with a novel CV component that interacts with the actin cytoskeleton. Analysis of radioiodinated brain CVs immunoprecipitated with a tubulin antibody revealed that all brain CVs contained tubulin. The mAb A-7C11 recognized a 40-kilodalton (kDa) polypeptide on the clathrin coat and immunoprecipitated one-quarter of the total brain CVs. The mAb S-11D9 reacted with a 44-kDa antigen and immunoprecipitated 25% of the CVs. This antigen (44 kDa) was present in synaptic vesicles and synaptosomal membrane as well. Moreover, this mAb (S-11D9) reacted with a polypeptide of 56 kDa detected only in synaptosomal membrane. A mAb (C-10B2) that reacted with one of the clathrin light chains (LCb) immunoprecipitated 90% of the brain CVs. One of the mAbs immunoprecipitated a CV subtype that displayed a reversed ratio of the clathrin LCs (LCa greater than LCb). Each of the mAbs yielded different immunofluorescent staining patterns of vesicles in culture cell types that included nerve growth factor-differentiated PC12 cells, neuroblastoma cells, and Madin Darby bovine kidney cells. The data suggest that in brain tissue there is a heterogeneous population of CVs with different polypeptide compositions and subcellular distributions and that each of these subtypes performs a different role in nerve cells.     

Non-Specific Detection of the Centrosomes by Antibodies Recognizing Phosphorylated ATM at Serine 1981

No abstract available.     

Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrP^Sc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.     

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na⁺/H⁺ antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P₃) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His₆ proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475–589), F2 (amino acids 590–667), F3 (amino acids 668–747), and F4 (amino acids 748–832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P₂ and PI(3,4,5)P₃ bound only to the NHE3 F1 fusion protein (amino acids 475–589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na⁺/H⁺ exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P₂ and PI(3,4,5)P₃ binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P₂ and PI(3,4,5)P₃ binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P₂ or PI(3,4,5)P₃ binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr⁵⁰¹–Arg⁵¹² and Arg⁵²⁰–Arg⁵⁵²) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.     

Immunocytochemical Detection of Anti-Hippocampal Antibodies in Alzheimer's Disease and Normal Cerebrospinal Fluid

Immunocytochemical staining was performed to investigate the presence of anti-hippocampal antibodies in cerebrospinal fluid (CSF) from patients with probable Alzheimer's disease (AD) (n = 19), aged normal controls (n = 9), and young normal controls (n = 10). Marked staining of neurons in the granule cell layer of the dentate gyrus and in pyramidal neurons in CA1-3 of the rat hippocampus was observed in 5 AD CSF samples (26%), 1 aged control sample (11%), and 1 young control sample (10%). These differences were not statistically significant. One of the immunoreactive AD CSF specimens also contained high concentrations of C5b-9, the membrane attack complex. The infrequent occurrence of anti-hippocampal antibodies in AD CSF, and the detection of similar immunoreactivity in control CSF specimens, suggest that these antibodies are unlikely to play a role in the neurodegenerative process in most individuals with AD. However, elevated C5b-9 concentration in an AD CSF specimen with marked immunoreactivity to hippocampal neurons suggests the possibility that anti-neuronal antibodies may contribute to complement activation in some AD patients.     

Artefacts in HPLC Detection of 3-Nitrotyrosine in Human Brain Tissue

Abstract: An HPLC method was used for quantification of 3-nitrotyrosine (3-NT) in human postmortem brain tissue. A peak with similar retention time to 3-NT was detected in brain tissue from patients with Parkinson's disease, Huntington's chorea, multiple system atrophy, and Alzheimer's disease but not in control tissue. The peak was lost on reduction with dithionite, a criterion often used to identify 3-NT. Tissue from the same neurodegenerative diseases was analysed by HPLC using a photodiode array detector in series with an amperometric electrochemical detector, but the peak was found not to be 3-NT. The absorbance spectrum, fragmentation pattern on mass spectroscopy, and electrochemical profile of this peak do not match authentic 3-NT. A search of the mass spectroscopy databases failed to reveal its identity. The presence of this closely eluting, dithionite-reducible peak could confound analysis of human tissues for 3-NT. In vitro experiments showed that high concentrations of peroxynitrite were needed to achieve detectable levels of 3-NT in human brain tissue.     

Development of Rabbit Monoclonal Antibodies for Detection of Alpha-Dystroglycan in Normal and Dystrophic Tissue

Alpha-dystroglycan requires a rare O-mannose glycan modification to form its binding epitope for extracellular matrix proteins such as laminin. This functional glycan is disrupted in a cohort of muscular dystrophies, the secondary dystroglycanopathies, and is abnormal in some metastatic cancers. The most commonly used reagent for detection of alpha-dystroglycan is mouse monoclonal antibody IIH6, but it requires the functional O-mannose structure for recognition. Therefore, the ability to detect alpha-dystroglycan protein in disease states where it lacks the full O-mannose glycan has been limited. To overcome this hurdle, rabbit monoclonal antibodies against the alpha-dystroglycan C-terminus were generated. The new antibodies, named 5–2, 29–5, and 45–3, detect alpha-dystroglycan from mouse, rat and pig skeletal muscle by Western blot and immunofluorescence. In a mouse model of fukutin-deficient dystroglycanopathy, all antibodies detected low molecular weight alpha-dystroglycan in disease samples demonstrating a loss of functional glycosylation. Alternately, in a porcine model of Becker muscular dystrophy, relative abundance of alpha-dystroglycan was decreased, consistent with a reduction in expression of the dystrophin-glycoprotein complex in affected muscle. Therefore, these new rabbit monoclonal antibodies are suitable reagents for alpha-dystroglycan core protein detection and will enhance dystroglycan-related studies.     

Preclinical Detection of Variant CJD and BSE Prions in Blood

The emergence of variant Creutzfeldt Jakob Disease (vCJD) is considered a likely consequence of human dietary exposure to Bovine Spongiform Encephalopathy (BSE) agent. More recently, secondary vCJD cases were identified in patients transfused with blood products prepared from apparently healthy donors who later went on to develop the disease. As there is no validated assay for detection of vCJD/BSE infected individuals the prevalence of the disease in the population remains uncertain. In that context, the risk of vCJD blood borne transmission is considered as a serious concern by health authorities. In this study, appropriate conditions and substrates for highly efficient and specific in vitro amplification of vCJD/BSE agent using Protein Misfolding Cyclic Amplification (PMCA) were first identified. This showed that whatever the origin (species) of the vCJD/BSE agent, the ovine Q₁₇₁ PrP substrates provided the best amplification performances. These results indicate that the homology of PrP amino-acid sequence between the seed and the substrate is not the crucial determinant of the vCJD agent propagation in vitro. The ability of this method to detect endogenous vCJD/BSE agent in the blood was then defined. In both sheep and primate models of the disease, the assay enabled the identification of infected individuals in the early preclinical stage of the incubation period. Finally, sample panels that included buffy coat from vCJD affected patients and healthy controls were tested blind. The assay identified three out of the four tested vCJD affected patients and no false positive was observed in 141 healthy controls. The negative results observed in one of the tested vCJD cases concurs with results reported by others using a different vCJD agent blood detection assay and raises the question of the potential absence of prionemia in certain patients.     

Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

Detection and Validation of QTL Affecting Bacterial Cold Water Disease Resistance in Rainbow Trout Using Restriction-Site Associated DNA Sequencing

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. Using microsatellite markers in a genome scan, we previously detected significant and suggestive QTL affecting phenotypic variation in survival following challenge with Flavobacterium psychrophilum, the causative agent of BCWD in rainbow trout. In this study, we performed selective genotyping of SNPs from restriction-site associated DNA (RAD) sequence data from two pedigreed families (2009070 and 2009196) to validate the major QTL from the previous work and to detect new QTL. The use of RAD SNPs in the genome scans increased the number of mapped markers from ~300 to ~5,000 per family. The significant QTL detected in the microsatellites scan on chromosome Omy8 in family 2009070 was validated explaining up to 58% of the phenotypic variance in that family, and in addition, a second QTL was also detected on Omy8. Two novel QTL on Omy11 and 14 were also detected, and the previously suggestive QTL on Omy1, 7 and 25 were also validated in family 2009070. In family 2009196, the microsatellite significant QTL on Omy6 and 12 were validated and a new QTL on Omy8 was detected, but none of the previously detected suggestive QTL were validated. The two Omy8 QTL from family 2009070 and the Omy12 QTL from family 2009196 were found to be co-localized with handling and confinement stress response QTL that our group has previously identified in a separate pedigreed family. With the currently available data we cannot determine if the co-localized QTL are the result of genes with pleiotropic effects or a mere physical proximity on the same chromosome segment. The genetic markers linked to BCWD resistance QTL were used to query the scaffolds of the rainbow trout reference genome assembly and the QTL-positive scaffold sequences were found to include 100 positional candidate genes. Several of the candidate genes located on or near the two Omy8 QTL detected in family 2009070 suggest potential linkages between stress response and the regulation of immune response in rainbow trout.     

Identification of Novel Genetic Loci Associated with Thyroid Peroxidase Antibodies and Clinical Thyroid Disease

Autoimmune thyroid diseases (AITD) are common, affecting 2-5% of the general population. Individuals with positive thyroid peroxidase antibodies (TPOAbs) have an increased risk of autoimmune hypothyroidism (Hashimoto''s thyroiditis), as well as autoimmune hyperthyroidism (Graves'' disease). As the possible causative genes of TPOAbs and AITD remain largely unknown, we performed GWAS meta-analyses in 18,297 individuals for TPOAb-positivity (1769 TPOAb-positives and 16,528 TPOAb-negatives) and in 12,353 individuals for TPOAb serum levels, with replication in 8,990 individuals. Significant associations (P<5×10⁻⁸) were detected at TPO-rs11675434, ATXN2-rs653178, and BACH2-rs10944479 for TPOAb-positivity, and at TPO-rs11675434, MAGI3-rs1230666, and KALRN-rs2010099 for TPOAb levels. Individual and combined effects (genetic risk scores) of these variants on (subclinical) hypo- and hyperthyroidism, goiter and thyroid cancer were studied. Individuals with a high genetic risk score had, besides an increased risk of TPOAb-positivity (OR: 2.18, 95% CI 1.68–2.81, P = 8.1×10⁻⁸), a higher risk of increased thyroid-stimulating hormone levels (OR: 1.51, 95% CI 1.26–1.82, P = 2.9×10⁻⁶), as well as a decreased risk of goiter (OR: 0.77, 95% CI 0.66–0.89, P = 6.5×10⁻⁴). The MAGI3 and BACH2 variants were associated with an increased risk of hyperthyroidism, which was replicated in an independent cohort of patients with Graves'' disease (OR: 1.37, 95% CI 1.22–1.54, P = 1.2×10⁻⁷ and OR: 1.25, 95% CI 1.12–1.39, P = 6.2×10⁻⁵). The MAGI3 variant was also associated with an increased risk of hypothyroidism (OR: 1.57, 95% CI 1.18–2.10, P = 1.9×10⁻³). This first GWAS meta-analysis for TPOAbs identified five newly associated loci, three of which were also associated with clinical thyroid disease. With these markers we identified a large subgroup in the general population with a substantially increased risk of TPOAbs. The results provide insight into why individuals with thyroid autoimmunity do or do not eventually develop thyroid disease, and these markers may therefore predict which TPOAb-positives are particularly at risk of developing clinical thyroid dysfunction.