Top Variants Research Articles

AAV capsid bioengineering in primary human retina models

Adeno-associated viral (AAV) vector-mediated retinal gene therapy is an active field of both pre-clinical as well as clinical research. As with other gene therapy clinical targets, novel bioengineered AAV variants developed by directed evolution or rational design to possess unique desirable properties, are entering retinal gene therapy translational programs. However, it is becoming increasingly evident that predictive preclinical models are required to develop and functionally validate these novel AAVs prior to clinical studies. To investigate if, and to what extent, primary retinal explant culture could be used for AAV capsid development, this study performed a large high-throughput screen of 51 existing AAV capsids in primary human retina explants and other models of the human retina. Furthermore, we applied transgene expression-based directed evolution to develop novel capsids for more efficient transduction of primary human retina cells and compared the top variants to the strongest existing benchmarks identified in the screening described above. A direct side-by-side comparison of the newly developed capsids in four different in vitro and ex vivo model systems of the human retina allowed us to identify novel AAV variants capable of high transgene expression in primary human retina cells.

Sex‐specific genetic architecture of multiple domains of cognition among older adults

AbstractBackgroundGenetic analyses of cognitive endophenotypes have led to discoveries of novel loci contributing to Alzheimer’s disease (AD) risk. Sex differences are present in cognitive trajectories in aging and AD, and these may vary across cognitive domain. However, genetic drivers that may contribute to sex differences in cognitive trajectories have yet to be explored. Thus, we sought to investigate the sex‐specific genetic architecture of cognition.MethodWe leveraged 10 cohorts of cognitive aging and AD to complete this sex‐aware genetic study (N = 31,800; mean age = 73 yrs.; 55% female). Harmonized cognitive scores for memory, executive functioning, and language were derived using confirmatory factor analysis models. We calculated change in cognitive scores over time using a mixed effects model, to facilitate analysis on cognitive decline. We performed GWAS of baseline score and of estimated rate of decline in each domain and in each cohort separately among non‐Hispanic white (NHW) individuals, adjusting for baseline age and genetic principal components. Then we meta‐analyzed the results.ResultIn addition to the well‐characterized APOE locus, we identified a genome‐wide significant chromosome 2 locus that was associated with language decline among NHW women (rs13387871: MAF = 0.20; βwomen = 2.97×10−3; Pwomen = 2.65×10−9), but not among male counterparts (βmen = ‐3.14×10−4, Pmen = 0.60). This locus contains multiple eQTLs for VRK2, a serine/threonine kinase that has been previously linked to neuropsychiatric disorders, including schizophrenia. Furthermore, the top variant in this locus (rs13387871) was nominally significant for memory decline (βwomen = 1.74×10−3, Pwomen = 0.01) and for executive functioning decline (βwomen = 7.58×10−4, Pwomen = 0.02) in meta‐analyses among NHW women.ConclusionOur genetic analysis suggests that there may be some genetic drivers of language performance that differ by sex, and that these drivers may be shared to an extent across domains. Our future sex‐aware meta‐analyses will also include 1) non‐Hispanic black (NHB) within ancestry (N = 4,200), 2) cross‐ancestry (NHW + NHB), 3) diagnosis‐stratified, and 4) analysis of X‐chromosome. Planned follow‐up analyses will include gene‐set analyses, heritability tests, and genetic correlation tests. Through our preliminary analysis, we identified a promising locus for further exploration, and this is the first of many steps in elucidating the sex‐specific genetic architecture of cognition across ancestry groups.

Amyloid β Induces Lipid Droplet‐Mediated Microglial Dysfunction in Alzheimer’s Disease

AbstractBackgroundSeveral microglia‐expressed genes have emerged as top risk variants for Alzheimer’s disease (AD) that are involved in lipid droplet (LD) formation and processing. Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD‐risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. LD accumulation in aging (Nat Neurosci. 23(2),194‐208, 2020) appears to increase microglial inflammatory responses, while inflammatory challenges increase LD formation, thereby potentially setting up a feed‐forward injury that could contribute to the relentless progression in AD.MethodLD formation is dependent upon age and disease progression, and is more prominent in the hippocampus in brains from human patients and the AD mouse model 5xFAD. Despite variability in LD load between microglia from male versus female animals and between cells from different brain regions, LD‐laden microglia exhibited a deficit in amyloid‐beta (Aβ) phagocytosis evaluated using AβpH ‐ a human Aβ1‐42 analog that exhibits fluorescence upon lysosomal internalization (Chem Sci. 12, 10901‐10918, 2021). Extensive unbiased lipidomic and metabolomic profiling in microglia identified a substantial decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TAGs) as the key metabolic transition underlying LD formation.ResultWe show that microglia form LDs upon exposure to Aβ, and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. Mechanistically, Aβ alone is sufficient to induce LD formation in microglia and that this conversion requires a specific metabolic conversion of FFAs to TAGs (the building blocks of LDs). This conversion is likely protective for the brain and microglia, as we recently showed that long‐chain saturated FFAs can be neurotoxic (Nature 599,102‐107, 2021), but it is not without cost for microglial phagocytic abilities. We demonstrate that a key enzyme for the conversion of FFAs to TAGs, promotes microglial LD formation, is increased in microglia from 5xFAD and human AD brains, and that inhibiting it improved microglial uptake of Aβ.ConclusionThese findings identify a new lipid‐mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

X‐chromosome wide association study for Alzheimer’s disease

AbstractBackgroundAlthough the X‐chromosome represents about 5% of the genome, it has been excluded from the major genome‐wide association studies on Alzheimer’s Disease (AD) due to its genetic specificities. We performed an in‐depth X‐Chromosome‐Wide Association Study (XWAS) to better characterize the genetic landscape of AD.MethodThe XWAS is based on 49,333 clinically diagnosed cases and 68,882 controls (including 31,314 female cases and 39,407 female controls) of European ancestry from 29 studies from the International Genomics of Alzheimer’s Project (IGAP) and the European Alzheimer & Dementia Biobank (EADB) consortia. 45,993 samples were imputed with the 1000G reference panel and 72,220 samples were imputed with the TOPMed panel.We considered three approaches to take into account the different inactivation scenarios on the X‐chromosome: 1) random X‐chromosome inactivation (r‐XCI) in females, equivalent to an additive genetic model with males coded as homozygous females; 2) skewed X‐chromosome inactivation in females, where a dominance effect was added to the r‐XCI model to capture the inactivation skewedness; 3) escape X‐chromosome inactivation in females, equivalent to an additive model with males coded as hemizygous. For each approach, we performed a single variant analysis with a meta‐analysis of the 29 studies, followed by a gene‐based analysis.ResultWe analyzed 404,688 variants, including 225,045 common variants (Minor Allele Frequency [MAF] > 1%). No genome‐wide significant signals were found in either approach, but three suggestive signals (p‐value < 1×10‐5) were identified in the r‐XCI approach. In one of these loci, we detected an X‐chromosome‐wide significant gene with the gene‐based analysis (p‐value = 5.45×10‐5). The top variant of this locus shows a similar effect in males and females (odds ratio [OR] = 0.93, 95% confidence interval [CI] = 0.90–0.96, p‐value = 8.63 × 10−6, and MAF = 32%, with OR[CI] in females = 0.86[0.79‐0.94], and OR[CI] in males = 0.92 [0.87‐0.97]).ConclusionWe identified promising genetic signals for AD on the X‐chromosome. To increase power, we are currently updating the imputation with TOPMed for all the studies and extending the analyses to large European biobanks.

Unveiling the Hidden Power of Uromodulin: A Promising Potential Biomarker for Kidney Diseases.

Uromodulin, also known as Tamm-Horsfall protein, represents the predominant urinary protein in healthy individuals. Over the years, studies have revealed compelling associations between urinary and serum concentrations of uromodulin and various parameters, encompassing kidney function, graft survival, cardiovascular disease, glucose metabolism, and overall mortality. Consequently, there has been a growing interest in uromodulin as a novel and effective biomarker with potential applications in diverse clinical settings. Reduced urinary uromodulin levels have been linked to an elevated risk of acute kidney injury (AKI) following cardiac surgery. In the context of chronic kidney disease (CKD) of different etiologies, urinary uromodulin levels tend to decrease significantly and are strongly correlated with variations in estimated glomerular filtration rate. The presence of uromodulin in the serum, attributable to basolateral epithelial cell leakage in the thick ascending limb, has been observed. This serum uromodulin level is closely associated with kidney function and histological severity, suggesting its potential as a biomarker capable of reflecting disease severity across a spectrum of kidney disorders. The UMOD gene has emerged as a prominent locus linked to kidney function parameters and CKD risk within the general population. Extensive research in multiple disciplines has underscored the biological significance of the top UMOD gene variants, which have also been associated with hypertension and kidney stones, thus highlighting the diverse and significant impact of uromodulin on kidney-related conditions. UMOD gene mutations are implicated in uromodulin-associated kidney disease, while polymorphisms in the UMOD gene show a significant association with CKD. In conclusion, uromodulin holds great promise as an informative biomarker, providing valuable insights into kidney function and disease progression in various clinical scenarios. The identification of UMOD gene variants further strengthens its relevance as a potential target for better understanding kidney-related pathologies and devising novel therapeutic strategies. Future investigations into the roles of uromodulin and regulatory mechanisms are likely to yield even more profound implications for kidney disease diagnosis, risk assessment, and management.

Developing flexible models for genetic evaluations in smallholder crossbred dairy farms

The productivity of smallholder dairy farms is very low in developing countries. Important genetic gains could be realized using genomic selection, but genetic evaluations need to be tailored for lack of pedigree information and very small farm sizes. To accommodate this situation, we propose a flexible Bayesian model for the genetic evaluation of milk yield, which allows us to simultaneously account for nongenetic random effects for farms and varying SNP variance (BayesR model). First, we used simulations based on real genotype data from Indian crossbred dairy cattle to demonstrate that the proposed model can separate the true genetic and nongenetic parameters even for small farm sizes (2 cows on average) although with high standard errors in scenarios with low heritability. The accuracy of genomic genetic evaluation increased until farm size was approximately 5. We then applied the model to real data from 4,655 crossbred cows with 106,109 monthly test day milk records and 689,750 autosomal SNPs. We estimated a heritability of 0.16 (0.04) for milk yield and using cross-validation, a genomic estimated breeding value (GEBV) accuracy of 0.45 and bias (regression of phenotype on GEBV) of 1.04 (0.26). Estimated genetic parameters were very similar using BayesR, BayesC, and genomic BLUP approaches. Candidate genes near the top variants, IMMP2L and ARHGEF2, have been previously associated with milk protein composition, mastitis resistance, and milk cholesterol content. The estimated heritability and GEBV accuracy for milk yield are much lower than those from intensive or pasture-based systems in many countries. Further increases in the number of phenotyped and genotyped animals in farms with at least 2 cows (preferably 3-5, to allow for dropout of cows) are needed to improve the estimation of genetic effects in these smallholder dairy farms.

A de novo mutation of ADAMTS8 in a patient with Wiedemann–Steiner syndrome

BackgroundWiedemann–Steiner syndrome (WDSTS) is a rare autosomal dominant disorder caused by mutations in the KMT2A gene and is usually characterized by hairy elbows, short stature, developmental delay, intellectual disability and obvious facial dysmorphism.Case presentationHere, we report a 5-year-old girl with clinical features similar to WDSTS, including postnatal growth delay, retarded intellectual development, and ocular hypertelorism. Through whole-exome sequencing (WES), a frameshift variant of KMT2A was found in the patient but not in her parents’ genomic DNA. By bioinformatics analysis, the KMT2A variant was demonstrated to be the top candidate pathogenic variant for the clinical phenotype consistent with WDSTS. Moreover, a duplication of exon 1 in ADAMTS8 (belonging to the zinc metalloproteinase family) was found in the genomic DNA of this patient, which may be responsible for the characteristics that are different from those of WDSTS, including early teething, rapid tooth replacement, and dysplastic enamel.ConclusionsFrom the above results, we propose that in our patient, the frameshift variant in KMT2A is the main reason for the WDSTS phenotype, and the unreported mutation in ADAMTS8 may be the candidate reason for other characteristics that are different from those of WDSTS. Therefore, this study not only provides a new KMT2A variant associated with WDSTS but is also a reminder that combined mutations may be present in a case with more characteristics than those seen in WDSTS.

A common variant that alters SUN1 degradation associates with hepatic steatosis and metabolic traits in multiple cohorts

Non-alcoholic fatty liver disease (NAFLD), and its progressive form steatohepatitis (NASH), represent a genetically and phenotypically diverse entity for which there is no approved therapy, making it imperative to define the spectrum of pathways contributing to its pathogenesis. Rare variants in genes encoding nuclear envelope proteins cause lipodystrophy with early-onset NAFLD/NASH; we hypothesized that common variants in nuclear envelope-related genes might also contribute to hepatic steatosis and NAFLD. Using hepatic steatosis as the outcome of interest, we performed an association meta-analysis of nuclear envelope-related coding variants in three large discovery cohorts (N >120,000 participants), followed by phenotype association studies in large validation cohorts (N >600,000) and functional testing of the top steatosis-associated variant in cell culture. A common protein-coding variant, rs6461378 (SUN1 H118Y), was the top steatosis-associated variant in our association meta-analysis (p <0.001). In ancestrally distinct validation cohorts, rs6461378 associated with histologic NAFLD and with NAFLD-related metabolic traits including increased serum fatty acids, type 2 diabetes, hypertension, cardiovascular disease, and decreased HDL. SUN1 H118Y was subject to increased proteasomal degradation relative to wild-type SUN1 in cells, and SUN1 H118Y-expressing cells exhibited insulin resistance and increased lipid accumulation. Collectively, these data support a potential causal role for the common SUN1 variant rs6461378 in NAFLD and metabolic disease. Non-alcoholic fatty liver disease (NAFLD), with an estimated global prevalence of nearly 30%, is a growing cause of morbidity and mortality for which there is no approved pharmacologic therapy. Our data provide a rationale for broadening current concepts of NAFLD genetics and pathophysiology to include the nuclear envelope, and particularly Sad1 and UNC84 domain containing 1 (SUN1), as novel contributors to this common liver disease. Furthermore, if future studies confirm causality of the common SUN1 H118Y variant, it has the potential to become a broadly relevant therapeutic target in NAFLD and metabolic disease.

X-Chromosome Association Study in Latin American Cohorts Identifies New Loci in Parkinson's Disease.

Sex differences in Parkinson's disease (PD) risk are well-known. However, the role of sex chromosomes in the development and progression of PD is still unclear. The objective of this study was to perform the first X-chromosome-wide association study for PD risk in a Latin American cohort. We used data from three admixed cohorts: (1) Latin American Research consortium on the Genetics of Parkinson's Disease (n = 1504) as discover cohort, and (2) Latino cohort from International Parkinson Disease Genomics Consortium (n = 155) and (3) Bambui Aging cohort (n = 1442) as replication cohorts. We also developed an X-chromosome framework specifically designed for admixed populations. We identified eight linkage disequilibrium regions associated with PD. We replicated one of these regions (top variant rs525496; discovery odds ratio [95% confidence interval]: 0.60 [0.478-0.77], P = 3.13 × 10-5 replication odds ratio: 0.60 [0.37-0.98], P = 0.04). rs5525496 is associated with multiple expression quantitative trait loci in brain and non-brain tissues, including RAB9B, H2BFM, TSMB15B, and GLRA4, but colocalization analysis suggests that rs5525496 may not mediate risk by expression of these genes. We also replicated a previous X-chromosome-wide association study finding (rs28602900), showing that this variant is associated with PD in non-European populations. Our results reinforce the importance of including X-chromosome and diverse populations in genetic studies. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Assessment of relationships between bullous pemphigoid and neurological diseases: A bidirectional two-sample Mendelian randomization study.

Bullous pemphigoid (BP) is the most prevalent autoimmune vesiculobullous skin illness that tends to affect the elderly. Growing evidence has hinted a correlation between BP and neurological diseases. However, existing observational studies contained inconsistent results, and the causality and direction of their relationship remain poorly understood. To assess the causal relationship between BP and neurological disorders, including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD), and stroke. A bidirectional two-sample Mendelian randomization (MR) adopted independent top genetic variants as instruments from the largest accessible genome-wide association studies (GWASs), with BP (n = 218 348), PD (n = 482 730), AD (n = 63 926), stroke (n = 446 696), and MS (n = 115 803). Inverse variance weighted (IVW), MR-Egger, weighted mode methods, weighted median, and simple mode were performed to explore the causal association. Multiple sensitivity analyses, MR-Pleiotropy Residual Sum and Outlier (PRESSO) was used to evaluate horizontal pleiotropy and remove outliers. With close-to-zero effect estimates, no causal impact of BP on the risk of the four neurological diseases was discovered. However, we found that MS was positively correlated with higher odds of BP (OR = 1.220, 95% CI: 1.058-1.408, p = 0.006), while no causal associations were observed between PD (OR = 0.821, 95% CI: 0.616-1.093, p = 0.176), AD (OR = 1.066, 95% CI: 0.873-1.358, p = 0.603), stroke (OR = 0.911, 95% CI: 0.485-1.713, p = 0.773) and odds of BP. In summary, no causal impact of BP on the risk of PD, AD, MS and stroke was detected in our MR analysis. However, reverse MR analysis identified that only MS was positively correlated with higher odds of BP, but not PD, AD and stroke.

Design Optimization and Analysis of Tubing Head Spool for Surface Wellheads are used in the Oil and Gas Industry

The Tubing Head serves as the uppermost spool in a surface wellhead and is typically positioned above the production casing, which is the final string of pipe suspended within the well. It plays a vital role in various functions from well drilling to initial oil production. Tubing head spools have been employed in conventional surface wellheads for many years. However, the current design of the spool is characterized by its significant weight and bulkiness. While this design has proven its effectiveness in the field, there is a need for improvements in terms of size and shape. The primary objective of this study is to reduce the weight and modify the size and shape of the tubing head spool, specifically the 13-5/8”-10K Bottom X 7-1/16”-10K Top variant, utilizing Solidworks modelling and simulation techniques. By leveraging these tools, we aim to optimize the design, achieve weight reduction, and enhance the overall performance of the tubing head spool. The optimization process begins with a comprehensive analysis of the existing tubing head spool design to identify potential areas for improvement. This analysis includes evaluating the stress distribution, pressure containment capabilities, and material utilization of the spool. Various optimization techniques, such as finite element analysis (FEA) are employed to simulate and analyse different design scenarios. We successfully, achieved weight reduction of 5.16% in raw forging and 14.45% in finish part.

1469-P: Gene-Level Gene–Mediterranean Diet Interaction Detection Influencing Inflammatory and Glycemic Biomarkers in 137,000 UK Biobank Participants

Adherence to a Mediterranean diet pattern strongly reduces type 2 diabetes risk on average, but there is substantial heterogeneity in the long-term glycemic and inflammatory response to dietary changes, likely due in part to genetic factors. Using a genome-wide interaction association analysis in the UK Biobank (UKB) cohort, we sought to prioritize and characterize genes contributing to this heterogeneity in the Mediterranean diet-risk factor relationship. We studied an unrelated, multi-ancestry UKB subset who completed at least one 24-hour diet questionnaire, had available glycemic (hemoglobin A1c; HbA1c) and inflammatory (high-sensitivity C-reactive protein; hsCRP) biomarkers measured in blood, and were free of major disease at baseline (N = 136,880). A nine-item, point-based Mediterranean diet score (MDS) based on food group and nutrient intakes was calculated. We conducted a series of genome-wide interaction studies (GWIS) to test for significant variant-diet interactions: one for the MDS itself and one for each sub-component. Variant-level results were aggregated by gene using the MAGMA tool. Gene-level MDS interaction results revealed two genes with false discovery rate &amp;lt; 0.1 for HbA1c: LRRC24 (p = 3.2×10−6) and CCDC40 (p = 3.3×10−6). Gene-level sub-component-specific analysis showed that these two interactions were primarily driven by alcohol and fish intake, respectively, and further prioritized an interaction between intake of nuts and LIN9 gene variants impacting HbA1c (p = 8.8×10−8). Follow-up analysis at the top variant in LIN9, rs9729447, showed a substantial attenuation of the negative nut intake-HbA1c association in major allele carriers and revealed a stronger interaction effect size in individuals who completed at least two dietary questionnaires. Our GWIS analysis suggests that there are locus-specific interactors with Mediterranean diet patterns influencing glycemic and inflammatory health. Disclosure K.E.Westerman: None. J.B.Meigs: Consultant; Quest Diagnostics. A.Manning: None. Funding National Institutes of Health (K01DK133637)

Association between genomic alterations and response of triple-negative breast cancers (TNBC) to talimogene laherparepvec (TVEC) in combination with neoadjuvant chemotherapy (NACT).

598 Background: TVEC is an engineered oncolytic virus (OVs) approved for the treatment of melanoma. We published a phase 1/2 trial combining TVEC with NACT in early stage TNBC demonstrating increased pathologic complete response (pCR) compared to expected rates with NACT. RNAseq was performed on 37 pretreatment samples from enrolled patients to identify potential predictive genomic alterations. Methods: Transcriptomic RNA sequencing analysis was performed on macrodissected tumor tissue through the Moffitt Molecular Genomics core using Illumina Truseq RNA exome kits. Read adapters were detected using BBMerge (v37.88) and removed with cutadapt (v1.8.1). For gene expression analysis, processed raw reads were aligned to human genome HG19 using STAR (v2.5.3a). Gene expression was evaluated as read count at gene level with HTSeq (v0.6.1) and Gencode gene model v19. Gene expression data were normalized and differential expression between experimental groups were evaluated using DEseq2 (v 1.38.2). Ensembl was used for SNP data search. Results: Tumor mutation burden was not significantly different between pCR and non-pCR samples (median 227 vs 222 mut/MB, p = .11). Top 5 upregulated non-immunoglobulin genes associated with pCR in baseline samples were MYBL1, DNAH8, NR2E1, FGFR2, and SLC12A1 (FDR &lt; 1%). A single nucleotide polymorphism (SNP) of the EIF2A gene (c.C290G, p.T97S) was the top variant associated with response present in 21/37 samples (non-pCR = 17/21 vs. pCR = 4/21, FDR = &lt; 1%). This exon 4 coding region SNP is present in 17% of Black, 35% of White, and 46% of Asian individuals but its biologic or clinical significance is unknown. Lower EIF2A pathway activation was associated with inferior response to therapy. Additional data will be presented at the meeting. Conclusions: Genomic alterations including a EIF2A SNP were correlated with response to OV plus NAC in TNBC. EIF2A is an important mediator of cellular responses to OV and may play a role in susceptibility to oncolysis. Additional investigation and validation of these variations in experimental models of OV susceptibility may provide predictive biomarkers for TNBC patients treated with OV plus NAC therapy. Clinical trial information: NCT02779855 .

Polygenic risk scores point toward potential genetic mechanisms of type 2 myocardial infarction in people with HIV

BackgroundPeople with human immunodeficiency virus (HIV) infection (PWH) are at higher risk of myocardial infarction (MI) than those without HIV. About half of MIs in PWH are type 2 (T2MI), resulting from mismatch between myocardial oxygen supply and demand, in contrast to type 1 MI (T1MI), which is due to primary plaque rupture or coronary thrombosis. Despite worse survival and rising incidence in the general population, evidence-based treatment recommendations for T2MI are lacking. We used polygenic risk scores (PRS) to explore genetic mechanisms of T2MI compared to T1MI in PWH. MethodsWe derived 115 PRS for MI-related traits in 9541 PWH enrolled in the Centers for AIDS Research Network of Integrated Clinical Systems cohort with adjudicated T1MI and T2MI. We applied multivariate logistic regression analyses to determine the association with T1MI and T2MI. Based on initial findings, we performed gene set enrichment analysis of the top variants composing PRS associated with T2MI. ResultsWe found that T1MI was strongly associated with PRS for cardiovascular disease, lipid profiles, and metabolic traits. In contrast, PRS for alcohol dependence and cholecystitis, significantly enriched in energy metabolism pathways, were predictive of T2MI risk. The association remained after the adjustment for actual alcohol consumption. ConclusionsWe demonstrate distinct genetic traits associated with T1MI and T2MI among PWH further highlighting their etiological differences and supporting the role of energy regulation in T2MI pathogenesis.

The role of M1 to M2 macrophage polarization in the etiology of idiopathic gastroparesis: GWAS perspective

Abstract Gastroparesis is a serious medical condition characterized by delayed gastric emptying. An innate immune dysregulation and injury to the interstitial cells of Cajal and other components of the enteric nervous system is likely central to the pathogenesis of gastroparesis. We have done a GWAS comparing idiopathic and diabetic cases with controls as well as contrasting idiopathic versus diabetic cases. We report a novel genetic association of SLC15A4 locus with idiopathic gastroparesis. This signal is driven by multiple variants with top missense variant SLC15A4:NM145648:exon2:c.T716C:p.V239A, rs33990080. Altogether, among EUR cohort we report 69 carriers out of 214 versus controls 165 out of 896 (MAF: cases 0.18; MAF: controls 0.09) and OR: 1.9 (p-value logistic model 10^−6). The significant effect persists when idiopathic cases are compared with diabetic cases as the MAF of diabetic cases (0.10) is comparable to that of population controls (0.09). Solute carrier family 15 (SLC15) A4 is a lysosome-resident amino acid/oligopeptide transporter that is preferentially expressed in immune cells. It is required for TLR7/9-dependent type I interferon production and plays a critical role in the pathogenesis of lupus and colitis. SLC15A4 was shown to mediate M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress. Previous reports have shown that when the anti-inflammatory M2 macrophages are switched to pro-inflammatory M1 macrophages, delayed GE was evident in animal models. Current genetic findings suggest that a mechanism directly connected to macrophage polarization may be implicated in a subset of idiopathic gastroparesis patients.

Abstract 2058: Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia

Abstract Acute Lymphoblastic Leukaemia (ALL) patients experience relapse despite having a good prognosis; approximately 90% of children and 75% to 85% of adolescents and young adults survive for five years. Chemotherapy resistance and a poor prognosis are linked to relapsed acute lymphoblastic Leukaemia (R-ALL). Evidence suggests that the cytosolic 5 nucleotidase II (NT5C2) gene mutations play a crucial role in the emergence of recurrent ALL; The early R-ALL is usually accompanied by 6-MP resistance, which is typically brought on by gain-of-function mutations impacting NT5C2-driven mutation in relapse mutations by different mechanisms is still unknown. The study aims to identify novel top missense variants of NT5C2 that play a crucial role in the resistance of chemotherapy drugs, altered biological pathways and their impact on the treatment of relapse leukaemia. NT5C2 (5'-Nucleotidase, Cytosolic II) is a Protein Coding gene. This gene induces resistance to chemotherapeutic drug 6 mercaptopurine Which causes leukaemia cell growth and leukaemia-initiating cell activity. The NT5C2 gene variants are extracted from Genome Aggregation Database (gnomAD) and ExAC. The extracted data contained 2280 variants with chromosome number, position reference allele, and altered allele and these were functionally annotated using the ANNOVAR tool. The variants are screened through various computational tools to narrow down based on non-synonymous and stopgain mutations in the exonic region. The predicted 519 variants narrow down by scoring GERP (&amp;gt;2.0) and CADD PHRED (cut-off score &amp;gt;10), ClinVar, SIFT score, Polyphen, PhyloP, PhastCons, LRT score predictions and which indicates the top 10% deleterious variants including reported and unknown variants. The SNPs, insertions, deletions, CNVs or structural variants of top missense variants on genes, transcripts, and protein sequence determined on Variants Effect Predictor on the ensemble. These variants were verified on dbSNP and Clinvar through their variant IDs. The functional prediction of these variants was made to detect their significant role in R-ALL. These findings examine the future role of NT5C2 mutations in the chemoresistance of relapse ALL. This may help to find a new therapeutic combination of drugs to treat relapse acute lymphoblastic leukaemia to improve overall survival. Citation Format: Sugunakar V, Obul Reddy Bandapalli, Ramita Sharma. Identifications of novel variants using system biology-based approaches in NT5C2, its association to relapse acute lymphoblastic leukaemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2058.

Directed evolution of angiotensin-converting enzyme 2 peptidase activity profiles for therapeutic applications.

Angiotensin-converting enzyme 2 (ACE2) has been investigated for its ability to beneficially modulate the angiotensin receptor (ATR) therapeutic axis to treat multiple human diseases. Its broad substrate scope and diverse physiological roles, however, limit its potential as a therapeutic agent. In this work, we address this limitation by establishing a yeast display-based liquid chromatography screen that enabled use of directed evolution to discover ACE2 variants that possess both wild-type or greater Ang-II hydrolytic activity and improved specificity toward Ang-II relative to the off-target peptide substrate Apelin-13. To obtain these results, we screened ACE2 active site libraries to reveal three substitution-tolerant positions (M360, T371, and Y510) that can be mutated to enhance ACE2's activity profile and followed up on these hits with focused double mutant libraries to further improve the enzyme. Relative to wild-type ACE2, our top variant (T371L/Y510Ile) displayed a sevenfold increase in Ang-II turnover number (kcat ), a sixfold diminished catalytic efficiency (kcat /Km ) on Apelin-13, and an overall decreased activity on other ACE2 substrates that were not directly assayed in the directed evolution screen. At physiologically relevant substrate concentrations, T371L/Y510Ile hydrolyzes as much or more Ang-II than wild-type ACE2 with concomitant Ang-II:Apelin-13 specificity improvements reaching 30-fold. Our efforts have delivered ATR axis-acting therapeutic candidates with relevance to both established and unexplored ACE2 therapeutic applications and provide a foundation for further ACE2 engineering efforts.

Genetic analyses of DNA repair pathway associated genes implicate new candidate cancer predisposing genes in ancestrally defined ovarian cancer cases.

Not all familial ovarian cancer (OC) cases are explained by pathogenic germline variants in known risk genes. A candidate gene approach involving DNA repair pathway genes was applied to identify rare recurring pathogenic variants in familial OC cases not associated with known OC risk genes from a population exhibiting genetic drift. Whole exome sequencing (WES) data of 15 OC cases from 13 families tested negative for pathogenic variants in known OC risk genes were investigated for candidate variants in 468 DNA repair pathway genes. Filtering and prioritization criteria were applied to WES data to select top candidates for further analyses. Candidates were genotyped in ancestry defined study groups of 214 familial and 998 sporadic OC or breast cancer (BC) cases and 1025 population-matched controls and screened for additional carriers in 605 population-matched OC cases. The candidate genes were also analyzed in WES data from 937 familial or sporadic OC cases of diverse ancestries. Top candidate variants in ERCC5, EXO1, FANCC, NEIL1 and NTHL1 were identified in 5/13 (39%) OC families. Collectively, candidate variants were identified in 7/435 (1.6%) sporadic OC cases and 1/566 (0.2%) sporadic BC cases versus 1/1025 (0.1%) controls. Additional carriers were identified in 6/605 (0.9%) OC cases. Tumour DNA from ERCC5, NEIL1 and NTHL1 variant carriers exhibited loss of the wild-type allele. Carriers of various candidate variants in these genes were identified in 31/937 (3.3%) OC cases of diverse ancestries versus 0-0.004% in cancer-free controls. The strategy of applying a candidate gene approach in a population exhibiting genetic drift identified new candidate OC predisposition variants in DNA repair pathway genes.

Identification of Novel, Replicable Genetic Risk Loci for Suicidal Thoughts and Behaviors Among US Military Veterans

Suicide is a leading cause of death; however, the molecular genetic basis of suicidal thoughts and behaviors (SITB) remains unknown. To identify novel, replicable genomic risk loci for SITB. This genome-wide association study included 633 778 US military veterans with and without SITB, as identified through electronic health records. GWAS was performed separately by ancestry, controlling for sex, age, and genetic substructure. Cross-ancestry risk loci were identified through meta-analysis. Study enrollment began in 2011 and is ongoing. Data were analyzed from November 2021 to August 2022. SITB. A total of 633 778 US military veterans were included in the analysis (57 152 [9%] female; 121 118 [19.1%] African ancestry, 8285 [1.3%] Asian ancestry, 452 767 [71.4%] European ancestry, and 51 608 [8.1%] Hispanic ancestry), including 121 211 individuals with SITB (19.1%). Meta-analysis identified more than 200 GWS (P < 5 × 10-8) cross-ancestry risk single-nucleotide variants for SITB concentrated in 7 regions on chromosomes 2, 6, 9, 11, 14, 16, and 18. Top single-nucleotide variants were largely intronic in nature; 5 were independently replicated in ISGC, including rs6557168 in ESR1, rs12808482 in DRD2, rs77641763 in EXD3, rs10671545 in DCC, and rs36006172 in TRAF3. Associations for FBXL19 and AC018880.2 were not replicated. Gene-based analyses implicated 24 additional GWS cross-ancestry risk genes, including FURIN, TSNARE1, and the NCAM1-TTC12-ANKK1-DRD2 gene cluster. Cross-ancestry enrichment analyses revealed significant enrichment for expression in brain and pituitary tissue, synapse and ubiquitination processes, amphetamine addiction, parathyroid hormone synthesis, axon guidance, and dopaminergic pathways. Seven other unique European ancestry-specific GWS loci were identified, 2 of which (POM121L2 and METTL15/LINC02758) were replicated. Two additional GWS ancestry-specific loci were identified within the African ancestry (PET112/GATB) and Hispanic ancestry (intergenic locus on chromosome 4) subsets, both of which were replicated. No GWS loci were identified within the Asian ancestry subset; however, significant enrichment was observed for axon guidance, cyclic adenosine monophosphate signaling, focal adhesion, glutamatergic synapse, and oxytocin signaling pathways across all ancestries. Within the European ancestry subset, genetic correlations (r > 0.75) were observed between the SITB phenotype and a suicide attempt-only phenotype, depression, and posttraumatic stress disorder. Additionally, polygenic risk score analyses revealed that the Million Veteran Program polygenic risk score had nominally significant main effects in 2 independent samples of veterans of European and African ancestry. The findings of this analysis may advance understanding of the molecular genetic basis of SITB and provide evidence for ESR1, DRD2, TRAF3, and DCC as cross-ancestry candidate risk genes. More work is needed to replicate these findings and to determine if and how these genes might impact clinical care.

Association of daytime napping frequency and schizophrenia: a bidirectional two-sample Mendelian randomization study

BackgroundThe bidirectional causal association between daytime napping frequency and schizophrenia is unclear.MethodsA bidirectional two-sample Mendelian randomization (MR) analysis was conducted with summary statistics of top genetic variants associated with daytime napping frequency and schizophrenia from genome-wide association studies (GWAS). The single nucleotide polymorphisms (SNPs) data of daytime napping frequency GWAS came from the UK Biobank (n = 452,633) and 23andMe study cohort (n = 541,333), while the schizophrenia GWAS came from the Psychiatric Genomics Consortium (PGC, 36,989 cases and 113,075 controls). The inverse variance weighted (IVW) analysis was the primary method, with the weighted median, MR-Robust Adjusted Profile Score (RAPS), Radial MR and MR-Pleiotropy Residual Sum Outlier (PRESSO) as sensitivity analysis.ResultsThe MR analysis showed a bidirectional causal relationship between more frequent daytime napping and the occurrence of schizophrenia, with the odds ratio (OR) for one-unit increase in napping category (never, sometimes, usually) on schizophrenia was 3.38 (95% confidence interval [CI]: 2.02–5.65, P = 3.58 × 10–6), and the beta for the occurrence of schizophrenia on daytime napping frequency was 0.0112 (95%CI: 0.0060–0.0163, P = 2.04 × 10–5). The sensitivity analysis obtained the same conclusions.ConclusionOur findings support the bidirectional causal association between more daytime napping frequency and schizophrenia, implying that daytime napping frequency is a potential intervention for the progression and treatment of schizophrenia.