Spatial Distribution of Missense Variants within Complement Proteins Associates with Age Related Macular Degeneration.

Purpose: Genetic variants in complement genes are associated with age-related macular degeneration (AMD). However, many rare variants have been identified in these genes, but have an unknown significance, and their impact on protein function and structure is still unknown. We set out to address this issue by evaluating the spatial placement and impact on protein structureof these variants by developing an analytical pipeline and applying it to the International AMD Genomics Consortium (IAMDGC) dataset (16,144 AMD cases, 17,832 controls). Methods: The IAMDGC dataset was imputed using the Haplotype Reference Consortium (HRC), leading to an improvement of over 30% more imputed variants, over the original 1000 Genomes imputation. Variants were extracted for the CFH , CFI , CFB , C9 , and C3 genes, and filtered for missense variants in solved protein structures. We evaluated these variants as to their placement in the three-dimensional structure of the protein (i.e. spatial proximity in the protein), as well as AMD association. We applied several pipelines to a) calculate spatial proximity to known AMD variants versus gnomAD variants, b) assess a variant's likelihood of causing protein destabilization via calculation of predicted free energy change (ddG) using Rosetta, and c) whole gene-based testing to test for statistical associations. Gene-based testing using seqMeta was performed using a) all variants b) variants near known AMD variants or c) with a ddG >|2|. Further, we applied a structural kernel adaptation of SKAT testing (POKEMON) to confirm the association of spatial distributions of missense variants to AMD. Finally, we used logistic regression on known AMD variants in CFI to identify variants leading to >50% reduction in protein expression from known AMD patient carriers of CFI variants compared to wild type (as determined by in vitro experiments) to determine the pipeline's robustness in identifying AMD-relevant variants. These results were compared to functional impact scores, ie CADD values > 10, which indicate if a variant may have a large functional impact genomewide, to determine if our metrics have better discriminative power than existing variant assessment methods. Once our pipeline had been validated, we then performed a priori selection of variants using this pipeline methodology, and tested AMD patient cell lines that carried those selected variants from the EUGENDA cohort (n=34). We investigated complement pathway protein expression in vitro , looking at multiple components of the complement factor pathway in patient carriers of bioinformatically identified variants. Results: Multiple variants were found with a ddG>|2| in each complement gene investigated. Gene-based tests using known and novel missense variants identified significant associations of the C3 , C9 , CFB , and CFH genes with AMD risk after controlling for age and sex (P=3.22×10 -5 ;7.58×10 -6 ;2.1×10 -3 ;1.2×10 -31 ). ddG filtering and SKAT-O tests indicate that missense variants that are predicted to destabilize the protein, in both CFI and CFH, are associated with AMD (P=CFH:0.05, CFI:0.01, threshold of 0.05 significance). Our structural kernel approach identified spatial associations for AMD risk within the protein structures for C3, C9, CFB, CFH, and CFI at a nominal p-value of 0.05. Both ddG and CADD scores were predictive of reduced CFI protein expression, with ROC curve analyses indicating ddG is a better predictor (AUCs of 0.76 and 0.69, respectively). A priori in vitro analysis of variants in all complement factor genes indicated that several variants identified via bioinformatics programs PathProx/POKEMON in our pipeline via in vitro experiments caused significant change in complement protein expression (P=0.04) in actual patient carriers of those variants, via ELISA testing of proteins in the complement factor pathway, and were previously unknown to contribute to AMD pathogenesis. Conclusion: We demonstrate for the first time that missense variants in complement genes cluster together spatially and are associated with AMD case/control status. Using this method, we can identify CFI and CFH variants of previously unknown significance that are predicted to destabilize the proteins. These variants, both in and outside spatial clusters, can predict in-vitro tested CFI protein expression changes, and we hypothesize the same is true for CFH . A priori identification of variants that impact gene expression allow for classification for previously classified as VUS. Further investigation is needed to validate the models for additional variants and to be applied to all AMD-associated genes.

Integrating Computational Approaches to Predict the Effect of Genetic Variants on Protein Stability in Retinal Degenerative Disease.

Protein function can be impacted by changes in protein structure stability, but determining which change has impact is complex. Stability can be affected by a large change in the tertiary (3D) structure of the protein or due to free-energy changes caused by single amino acid substitutions. Changes in the DNA sequence can have minor or major impact on protein stability, which can lead to disease. Inherited retinal degenerations are generally caused by single mutations which are mostly located in protein-coding regions, while age-related macular degeneration (AMD) is a complex disorder that can be influenced by some genetic variants impacting proteins involved in the disease, although not all AMD risk variants lead to amino acid changes. Here, we review ways that proteins may be affected, the identification and understanding of these changes, and how to identify causal changes that can be targeted to develop treatments to alleviate retinal degenerative disease.

Multi-ancestry genome-wide meta-analysis of 56,241 individuals identifies LRRC4C, LHX5-AS1 and nominates ancestry-specific loci PTPRK , GRB14 , and KIAA0825 as novel risk loci for Alzheimer's disease: the Alzheimer's Disease Genetics Consortium.

Limited ancestral diversity has impaired our ability to detect risk variants more prevalent in non-European ancestry groups in genome-wide association studies (GWAS). We constructed and analyzed a multi-ancestry GWAS dataset in the Alzheimer's Disease (AD) Genetics Consortium (ADGC) to test for novel shared and ancestry-specific AD susceptibility loci and evaluate underlying genetic architecture in 37,382 non-Hispanic White (NHW), 6,728 African American, 8,899 Hispanic (HIS), and 3,232 East Asian individuals, performing within-ancestry fixed-effects meta-analysis followed by a cross-ancestry random-effects meta-analysis. We identified 13 loci with cross-ancestry associations including known loci at/near CR1 , BIN1 , TREM2 , CD2AP , PTK2B , CLU , SHARPIN , MS4A6A , PICALM , ABCA7 , APOE and two novel loci not previously reported at 11p12 ( LRRC4C ) and 12q24.13 ( LHX5-AS1 ). Reflecting the power of diverse ancestry in GWAS, we observed the SHARPIN locus using 7.1% the sample size of the original discovering single-ancestry GWAS (n=788,989). We additionally identified three GWS ancestry-specific loci at/near ( PTPRK ( P =2.4×10 -8 ) and GRB14 ( P =1.7×10 -8 ) in HIS), and KIAA0825 ( P =2.9×10 -8 in NHW). Pathway analysis implicated multiple amyloid regulation pathways (strongest with P adjusted =1.6×10 -4 ) and the classical complement pathway ( P adjusted =1.3×10 -3 ). Genes at/near our novel loci have known roles in neuronal development ( LRRC4C, LHX5-AS1 , and PTPRK ) and insulin receptor activity regulation ( GRB14 ). These findings provide compelling support for using traditionally-underrepresented populations for gene discovery, even with smaller sample sizes.

Private well water testing promotion in pediatric preventive care: A randomized intervention study.

Over 43 million U.S. residents rely on private unregulated wells for their drinking water, raising public health concerns, particularly in regions like northern New England where widespread groundwater arsenic contamination is now recognized. Children are particularly vulnerable to adverse health effects from arsenic exposure. Despite AAP Guidelines, approaches to engage pediatric clinicians in promoting private well testing have not been previously described. We sought to determine the most effective practice approaches to achieve successful well water testing in routine pediatric care. 12 primary care clinics were block randomized to one of four study arms. Two intervention variables were assessed: (1) test results access (parent only vs. parent and clinic) and (2) follow up approaches (yes/no). Parents of children under 12 months using a private well were eligible. Prepaid water tests were provided. Primary outcome was parental water test completion. Eleven clinics successfully implemented processes identifying well users. 240 testing kits were dispensed. Completion rates averaged 29% (range 10 to 61%). The study arm with both clinic results access and staff follow up system was 2.3 times more likely to achieve test completion than other arms (95% CI 1.12-4.86, p = .03). Kit distribution by clinicians versus nursing staff, irrespective of study arm, had 2.4 times greater completion (95% CI 1.13-5.11, p = .02). Systematic drinking water source screening can be improved in pediatric care. Higher testing completion was found in practices randomized to reminders and structured follow up versus single visit discussion, but clinician involvement was the most predictive factor.

Non-literal understanding and psychosis: Metaphor comprehension in individuals with a diagnosis of schizophrenia.

Previous studies suggest that understanding of non-literal expressions, and in particular metaphors, can be impaired in people with schizophrenia; although it is not clear why. We explored metaphor comprehension capacity using a novel picture selection paradigm; we compared task performance between people with schizophrenia and healthy comparator subjects and we further examined the relationships between the ability to interpret figurative expressions non-literally and performance on a number of other cognitive tasks. Eye-tracking was used to examine task strategy. We showed that even when IQ, years of education, and capacities for theory of mind and associative learning are factored in as covariates, patients are significantly more likely to interpret metaphorical expressions literally, despite eye-tracking findings suggesting that patients are following the same interpretation strategy as healthy controls. Inhibitory control deficits are likely to be one of multiple factors contributing to the poorer performance of our schizophrenia group on the metaphor trials of the picture selection task.

Assessing data availability and quality within an electronic health record system through external validation against an external clinical data source.

BACKGROUND: Approximately 20% of deaths in the US each year are attributable to smoking, yet current practices in the recording of this health risk in electronic health records (EHRs) have not led to discernable changes in health outcomes. Several groups have developed algorithms for extracting smoking behaviors from clinical notes, but none of these approaches were assessed with external data to report on anticipated clinical performance. METHODS: Previously, we developed an informatics pipeline that extracts smoking status, pack year history, and cessation date from clinical notes. Here we report on the clinical implementation performance of our pipeline using 1,504 clinical notes matched to an external questionnaire. RESULTS: We found that 73% of available notes contained no smoking behavior information. The weighted Cohen's kappa between the external questionnaire and EHR smoking status was 0.62 (95% CI 0.56-0.69) for the clinical notes we were able to extract information from. The correlation between pack years reported by our pipeline and the external questionnaire was 0.39 on the 81 notes for which this information was present in both. We also assessed for lung cancer screening eligibility using notes from individuals identified as never smokers or smokers with pack year history extracted by our pipeline (n = 196). We found a positive predictive value of 85.4%, a negative predictive value of 83.8%, sensitivity of 63.1%, and specificity of 94.7%. CONCLUSIONS: We have demonstrated that our pipeline can extract smoking behaviors from unannotated EHR notes when the information is present. This information is reliable enough to identify patients most likely to be eligible for smoking related services. Ensuring capture of smoking information during clinical encounters should continue to be a high priority.

Building a tobacco user registry by extracting multiple smoking behaviors from clinical notes.

BACKGROUND: Usage of structured fields in Electronic Health Records (EHRs) to ascertain smoking history is important but fails in capturing the nuances of smoking behaviors. Knowledge of smoking behaviors, such as pack year history and most recent cessation date, allows care providers to select the best care plan for patients at risk of smoking attributable diseases. METHODS: We developed and evaluated a health informatics pipeline for identifying complete smoking history from clinical notes in EHRs. We utilized 758 patient-visit notes (from visits between 03/28/2016 and 04/04/2016) from our local EHR in addition to a public dataset of 502 clinical notes from the 2006 i2b2 Challenge to assess the performance of this pipeline. We used a machine-learning classifier to extract smoking status and a comprehensive set of text processing regular expressions to extract pack years and cessation date information from these clinical notes. RESULTS: We identified smoking status with an F1 score of 0.90 on both the i2b2 and local data sets. Regular expression identification of pack year history in the local test set was 91.7% sensitive and 95.2% specific, but due to variable context the pack year extraction was incomplete in 25% of cases, extracting packs per day or years smoked only. Regular expression identification of cessation date was 63.2% sensitive and 94.6% specific. CONCLUSIONS: Our work indicates that the development of an EHR-based Smokers' Registry containing information relating to smoking behaviors, not just status, from free-text clinical notes using an informatics pipeline is feasible. This pipeline is capable of functioning in external EHRs, reducing the amount of time and money needed at the institute-level to create a Smokers' Registry for improved identification of patient risk and eligibility for preventative and early detection services.

IFNgamma-producing, virus-specific CD8+ effector cells acquire the ability to produce IL-10 as a result of entry into the infected lung environment.

It has become clear that T cells with the potential to negatively regulate the immune response are normal constituents of the immune system. These cells often mediate their effects through the production of immunosuppressive factors. At present our understanding of how these cells are generated is limited. Here we report the presence of a population of IL-10-producing, virus-specific CD8+ T cells in the lungs of mice following acute respiratory infection. These cells were only found at minimal levels in the spleen and draining lymph node; instead they were restricted primarily to the infected lung tissue. A major finding from this study is demonstration that the ability to produce IL-10 can be acquired by IFNgamma-producing effector cells following entry into the infected lung. These studies suggest IL-10 production is the result of further differentiation of an antigen-specific CD8+ T cell that is governed by signals present in infected lung tissue.

Loss of function in virus-specific lung effector T cells is independent of infection.

Recently, several studies, including those with respiratory syncytial virus, mouse pneumovirus, and simian virus 5, have reported that virus-specific CD8+ effector cells entering the lung as a result of respiratory infection undergo significant loss of function. The impaired function in these cells has been proposed to be the result of infection-induced changes in the lung. Although virus-specific effects may contribute to regulation of T cells in the lung, the findings from this study provide evidence that the basal lung environment is sufficient to promote loss of function in effector cells. Loss of function occurs within 48 h of entry into the lung and is most evident in cells residing in the lung parenchyma. These findings suggest an additional paradigm for the immunoregulation of effector cells that enter the lung as a result of virus infection.

Altered function in CD8+ T cells following paramyxovirus infection of the respiratory tract.

For many respiratory pathogens, CD8+ T cells have been shown to play a critical role in clearance. However, there are still many unanswered questions with regard to the factors that promote the most efficacious immune response and the potential for immunoregulation of effector cells at the local site of infection. We have used infection of the respiratory tract with the model paramyxovirus simian virus 5 (SV5) to study CD8+ T-cell responses in the lung. For the present study, we report that over time a population of nonresponsive, virus-specific CD8+ T cells emerged in the lung, culminating in a lack of function in approximately 85% of cells specific for the immunodominant epitope from the viral matrix (M) protein by day 40 postinfection. Concurrent with the induction of nonresponsiveness, virus-specific cells that retained function at later times postinfection exhibited an increased requirement for CD8 engagement. This change was coupled with a nearly complete loss of functional phosphoprotein-specific cells, a response previously shown to be almost exclusively CD8 independent. These studies add to the growing evidence for immune dysregulation following viral infection of the respiratory tract.

Human helper T cell activation and differentiation is suppressed by porcine small intestinal submucosa.

A cell-free biomaterial derived from porcine small intestinal submucosa (SIS) has been used successfully in many models as a xenogeneic scaffolding material without generating immune-mediated inflammatory reactions. We investigated whether this absence of inflammation is due to the presence of porcine transforming growth factor beta (TGF-beta) activity found in SIS that may have immunosuppressive properties on helper T (Th) cell subset activation and differentiation. We used in vitro models for the generation of human Th1 and Th2 cells to investigate the influence of SIS. We found that SIS partially suppressed Th1 cell expansion and secretion of interleukin 12 (IL-12) and interferon gamma (IFN-gamma) in a TGF-beta-dependent manner, but Th1 cell expansion and IFN-gamma secretion could be fully overcome by addition of recombinant IL-12. The suppression by SIS of Th cell activation also involved the induction of Th cell apoptosis. In addition, SIS completely abolished the generation of Th2 cells in vitro, but this effect of SIS was not reversed by neutralizing TGF-beta antibodies. Our results indicate the presence in SIS of factors that can suppress Th cell activation through both the inhibition of IL-12 secretion and the induction of Th cell apoptosis. We established further that these factors include TGF-beta and at least one other factor.

Human fetal retinal pigment epithelium induces apoptosis in human T-cell line Jurkat which is independent from its expression of TRAIL.

PURPOSE: To evaluate whether human fetal retinal pigment epithelial (HFRPE) cells express TRAIL (tumor necrosis factor related apoptosis inducing ligand). The role of TRAIL in HFRPE induced apoptosis was evaluated. METHODS: Pure cultures of HFRPE cells were isolated. The expression of TRAIL protein and mRNA in non-activated and IFN-gamma activated HFRPE cells was evaluated with RT-PCR. The role of TRAIL in HFRPE induced apoptosis was assessed by incubating HFRPE cells with human T-cell leukemia line Jurkat (Jkt) in the presence or absence of neutralizing TRAIL antibodies. Cultures were pulsed with [(3)H]-thymidine to measure Jkt cell proliferation. The role of TRAIL was further examined by western blott evaluating the cleavage of caspases 8 and 10 in Jkt cells after their incubation with HFRPE cells. RESULTS: HFRPE cells expressed TRAIL mRNA. The expression of TRAIL mRNA and protein was up-regulated by IFN-gamma activation. However, anti-TRAIL antibodies were not able to prevent the HFRPE induced suppression of Jkt cell proliferation. The caspases 8 and 10 were also not cleaved in Jkt cells after their incubation with IFN-gamma activated HFRPE cells. CONCLUSIONS: Although HFRPE cells express TRAIL and its expression is upregulated by IFN-gamma activation, TRAIL is not involved in HFRPE induced apoptosis in Jkt cells. Currently the role of TRAIL in HFRPE cells is under investigation.