Genetic Association Studies in Host-Pathogen Interaction Analysis.

Studying host-pathogen interactions at a molecular level has always been technically challenging. Identifying the different biochemical and genetic pathways involved in the different stages of infection traditionally require complex molecular biology tools and often the use of costly animal models. In this chapter, we illustrate a complementary approach to address host-pathogen interactions, taking advantage of the natural interindividual genetic diversity. The application of genetic association studies allows us to identify alleles involved in infection progression or resistance. Thus, this strategy may be useful to unravel new molecular pathways underlying host-pathogen interactions. Here we present the general steps that might be followed to plan, execute, and analyze a population-based study in order to identify genetic variants affecting human exposition to pathogens.

The absence of seroconversion after exposition to hepatitis C virus is not related to KIR-HLA genotype combinations (GEHEP-012 study).

BACKGROUND & AIMS: It has been reported that specific killer-cell immunoglobulin-like receptors (KIRs) and HLA genotype combinations, such as KIR2DS4/HLA-C1 with presence of KIRDL2 or KIRDL3, homozygous KIRDL3/HLA-C1 and KIR3DL1/≥2HLA-Bw4, are strongly associated with the lack of active infection and seroconversion after exposition to hepatitis C virus (HCV). OBJECTIVE: To determine whether these KIR-HLA combinations are relevant factors involved in that phenotype. PATIENTS AND METHODS: In this retrospective case-control study, genotype data from a genome-wide association study previously performed on low susceptibility to HCV-infection carried out on 27 high-risk HCV-seronegative (HRSN) individuals and 743 chronically infected (CI) subjects were used. HLA alleles were imputed using R package HIBAG v1.2223 and KIR genotypes were imputed using the online resource KIR*IMP v1.2.0. RESULTS: It was possible to successfully impute at least one KIR-HLA genotype combination previously associated with the lack of infection and seroconversion after exposition to HCV in a total of 23 (85.2%) HRSN individuals and in 650 (87.5%) CI subjects. No KIR-HLA genotype combination analyzed was related to the HRSN condition. CONCLUSIONS: Our results suggest that those KIR-HLA genotype combinations are not relevant factors involved in the lack of infection and seroconversion after exposition to HCV. More studies will be needed to completely understand this phenotype.

A Functional Pipeline of Genome-Wide Association Data Leads to Midostaurin as a Repurposed Drug for Alzheimer's Disease.

Genome-wide association studies (GWAS) constitute a powerful tool to identify the different biochemical pathways associated with disease. This knowledge can be used to prioritize drugs targeting these routes, paving the road to clinical application. Here, we describe DAGGER (Drug Repositioning by Analysis of GWAS and Gene Expression in R), a straightforward pipeline to find currently approved drugs with repurposing potential. As a proof of concept, we analyzed a meta-GWAS of 1.6 × 107 single-nucleotide polymorphisms performed on Alzheimer's disease (AD). Our pipeline uses the Genotype-Tissue Expression (GTEx) and Drug Gene Interaction (DGI) databases for a rational prioritization of 22 druggable targets. Next, we performed a two-stage in vivo functional assay. We used a C. elegans humanized model over-expressing the Aß1-42 peptide. We assayed the five top-scoring candidate drugs, finding midostaurin, a multitarget protein kinase inhibitor, to be a protective drug. Next, 3xTg AD transgenic mice were used for a final evaluation of midostaurin's effect. Behavioral testing after three weeks of 20 mg/kg intraperitoneal treatment revealed a significant improvement in behavior, including locomotion, anxiety-like behavior, and new-place recognition. Altogether, we consider that our pipeline might be a useful tool for drug repurposing in complex diseases.

A metagenome-wide association study of HIV disease progression in HIV controllers.

Some HIV controllers experience immunologic progression with CD4+ T cell decline. We aimed to identify genetic factors associated with CD4+ T cell lost in HIV controllers. A total of 561 HIV controllers were included, 442 and 119 from the International HIV controllers Study Cohort and the Swiss HIV Cohort Study, respectively. No SNP or gene was associated with the long-term non-progressor HIV spontaneous control phenotype in the individual GWAS or in the meta-analysis. However, SNPs previously associated with natural HIV control linked to HLA-B (rs2395029 [p = 0.005; OR = 1.70], rs59440261 [p = 0.003; OR = 1.78]), MICA (rs112243036 [p = 0.011; OR = 1.45]), and PSORS1C1 loci (rs3815087 [p = 0.017; OR = 1.39]) showed nominal association with this phenotype. Genetic factors associated with the long-term HIV controllers without risk of immunologic progression are those previously related to the overall HIV controller phenotype.

Genetic Associations Between Modifiable Risk Factors and Alzheimer Disease.

Importance: An estimated 40% of dementia is potentially preventable by modifying 12 risk factors throughout the life course. However, robust evidence for most of these risk factors is lacking. Effective interventions should target risk factors in the causal pathway to dementia. Objective: To comprehensively disentangle potentially causal aspects of modifiable risk factors for Alzheimer disease (AD) to inspire new drug targeting and improved prevention. Design, Setting, and Participants: This genetic association study was conducted using 2-sample univariable and multivariable mendelian randomization. Independent genetic variants associated with modifiable risk factors were selected as instrumental variables from genomic consortia. Outcome data for AD were obtained from the European Alzheimer & Dementia Biobank (EADB), generated on August 31, 2021. Main analyses were conducted using the EADB clinically diagnosed end point data. All analyses were performed between April 12 and October 27, 2022. Exposures: Genetically determined modifiable risk factors. Main Outcomes and Measures: Odds ratios (ORs) and 95% CIs for AD were calculated per 1-unit change of genetically determined risk factors. Results: The EADB-diagnosed cohort included 39 106 participants with clinically diagnosed AD and 401 577 control participants without AD. The mean age ranged from 72 to 83 years for participants with AD and 51 to 80 years for control participants. Among participants with AD, 54% to 75% were female, and among control participants, 48% to 60% were female. Genetically determined high-density lipoprotein (HDL) cholesterol concentrations were associated with increased odds of AD (OR per 1-SD increase, 1.10 [95% CI, 1.05-1.16]). Genetically determined high systolic blood pressure was associated with increased risk of AD after adjusting for diastolic blood pressure (OR per 10-mm Hg increase, 1.22 [95% CI, 1.02-1.46]). In a second analysis to minimize bias due to sample overlap, the entire UK Biobank was excluded from the EADB consortium; odds for AD were similar for HDL cholesterol (OR per 1-SD unit increase, 1.08 [95% CI, 1.02-1.15]) and systolic blood pressure after adjusting for diastolic blood pressure (OR per 10-mm Hg increase, 1.23 [95% CI, 1.01-1.50]). Conclusions and Relevance: This genetic association study found novel genetic associations between high HDL cholesterol concentrations and high systolic blood pressure with higher risk of AD. These findings may inspire new drug targeting and improved prevention implementation.

Mendelian Randomisation Confirms the Role of Y-Chromosome Loss in Alzheimer's Disease Aetiopathogenesis in Men.

Mosaic loss of chromosome Y (mLOY) is a common ageing-related somatic event and has been previously associated with Alzheimer's disease (AD). However, mLOY estimation from genotype microarray data only reflects the mLOY degree of subjects at the moment of DNA sampling. Therefore, mLOY phenotype associations with AD can be severely age-confounded in the context of genome-wide association studies. Here, we applied Mendelian randomisation to construct an age-independent mLOY polygenic risk score (mloy-PRS) using 114 autosomal variants. The mloy-PRS instrument was associated with an 80% increase in mLOY risk per standard deviation unit (p = 4.22 × 10-20) and was orthogonal with age. We found that a higher genetic risk for mLOY was associated with faster progression to AD in men with mild cognitive impairment (hazard ratio (HR) = 1.23, p = 0.01). Importantly, mloy-PRS had no effect on AD conversion or risk in the female group, suggesting that these associations are caused by the inherent loss of the Y chromosome. Additionally, the blood mLOY phenotype in men was associated with increased cerebrospinal fluid levels of total tau and phosphorylated tau181 in subjects with mild cognitive impairment and dementia. Our results strongly suggest that mLOY is involved in AD pathogenesis.

Moderate exercise reveals the influence of ACTN3 R577X and ACE I/D polymorphisms on physical performance in non-athlete active subjects.

Genome variations contribute to the vast majority of interindividual differences and may decisively influence sports capability. This study was conceived as a means of finding out when exactly polymorphisms start being physically discriminative. The polymorphisms we studied were two of the best characterized ones: ACE I/D and ACTN3 R577X. These germline variants were determined in a cohort of 200 healthy volunteers from the university environment who underwent a series of physical evaluations that included a Cooper test, a 20-meter sprint test and a vertical jump test. Initially, no statistical association was found because the genetic effect was masked by those subjects with sedentary lifestyles. But when only physically active volunteers were considered, the ACE and ACTN3 genotypes were found to have an impact on heart rate after the Cooper test (p-value = 0.033 and 0.032 respectively) and ACTN3 was found to correlate with the total distance covered in the same test (p-value = 0.051). This can therefore be considered a paradigmatic example in which the environment might hide the genetic effect, with genotypic differences arising only upon training.

Genomic Characterization of Host Factors Related to SARS-CoV-2 Infection in People with Dementia and Control Populations: The GR@ACE/DEGESCO Study.

Emerging studies have suggested several chromosomal regions as potential host genetic factors involved in the susceptibility to SARS-CoV-2 infection and disease outcome. We nested a COVID-19 genome-wide association study using the GR@ACE/DEGESCO study, searching for susceptibility factors associated with COVID-19 disease. To this end, we compared 221 COVID-19 confirmed cases with 17,035 individuals in whom the COVID-19 disease status was unknown. Then, we performed a meta-analysis with the publicly available data from the COVID-19 Host Genetics Initiative. Because the APOE locus has been suggested as a potential modifier of COVID-19 disease, we added sensitivity analyses stratifying by dementia status or by disease severity. We confirmed the existence of the 3p21.31 region (LZTFL1, SLC6A20) implicated in the susceptibility to SARS-CoV-2 infection and TYK2 gene might be involved in COVID-19 severity. Nevertheless, no statistically significant association was observed in the COVID-19 fatal outcome or in the stratified analyses (dementia-only and non-dementia strata) for the APOE locus not supporting its involvement in SARS-CoV-2 pathobiology or COVID-19 prognosis.

The Genetic Research in Alzheimer Disease (GERALD) Initiative Finds rs9320913 as a Neural eQTL of lincRNA AL589740.1.

Alzheimer's disease is the most common cause of dementia worldwide, and longitudinal studies are crucial to find the factors affecting disease development. Here, we describe a novel initiative from southern Spain designed to contribute in the identification of the genetic component of the cognitive decline of Alzheimer's disease patients. The germline variant rs9320913 is a C>A substitution mapping within a gene desert. Although it has been previously associated to a higher educational achievement and increased fluid intelligence, its role on Alzheimer's disease risk and progression remains elusive. A total of 407 subjects were included in the study, comprising 153 Alzheimer disease patients and 254 healthy controls. We have explored the rs9320913 contribution to both Alzheimer disease risk and progression according to the Mini-Mental State Exams. We found that rs9320913 maps within a central nervous system lincRNA AL589740.1. eQTL results show that rs9320913 correlated with the brain-frontal cortex (beta = -0.15, p value = 0.057) and brain-spinal cord (beta of -0.23, p value = 0.037). We did not find rs9320913 to be associated to AD risk, although AA patients seemed to exhibit a less pronounced Mini-Mental State Exam score decline.

Common variants in Alzheimer's disease and risk stratification by polygenic risk scores.

Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease.

MAOB rs3027452 Modifies Mood Improvement After Tryptophan Supplementation.

PURPOSE: Tryptophan is the only precursor of serotonin, the hormone which helps regulate key human functions such as appetite, memory, mood, and sexual behavior. Connections have been identified between serotonin system dysfunction and the molecular etiology and treatment of mood disorders in a wide range of studies. Proposals have been put forward to co-administer tryptophan supplementation together with serotonin reuptake inhibitors in major depression patients, and also to exploit the sub-therapeutic depressive status in healthy populations. The reported responses, however, have been very dissimilar and this uneven effect may largely be explained by interindividual genetic differences. MATERIALS AND METHODS: We studied mood change in 138 healthy subjects using both Goldberg's General Health Questionnaire and the Profile of Mood States Questionnaire to determine the effects of a daily supplementation of 1g of tryptophan or placebo. Buccal DNA samples were provided and TPH1 (rs1800532), MAOA (rs3788862 and rs979605), MAOB (rs3027452), and COMT (rs6269 and rs4680) variants were genotyped. RESULTS: MAOB rs3027452 was equally associated with tryptophan supplementation efficacy in the depression subscales of both questionnaires (ΔT-Score.D; ΔT-Score.TMD and ΔPOMS.D p-values <0.01). CONCLUSION: Here we provide evidence that tryptophan supplementation has an uneven effect on mood improvement in the general population.

Long runs of homozygosity are associated with Alzheimer's disease.

Long runs of homozygosity (ROH) are contiguous stretches of homozygous genotypes, which are a footprint of inbreeding and recessive inheritance. The presence of recessive loci is suggested for Alzheimer's disease (AD); however, their search has been poorly assessed to date. To investigate homozygosity in AD, here we performed a fine-scale ROH analysis using 10 independent cohorts of European ancestry (11,919 AD cases and 9181 controls.) We detected an increase of homozygosity in AD cases compared to controls [ßAVROH (CI 95%) = 0.070 (0.037-0.104); P = 3.91 × 10-5; ßFROH (CI95%) = 0.043 (0.009-0.076); P = 0.013]. ROHs increasing the risk of AD (OR > 1) were significantly overrepresented compared to ROHs increasing protection (p < 2.20 × 10-16). A significant ROH association with AD risk was detected upstream the HS3ST1 locus (chr4:11,189,482‒11,305,456), (ß (CI 95%) = 1.09 (0.48 ‒ 1.48), p value = 9.03 × 10-4), previously related to AD. Next, to search for recessive candidate variants in ROHs, we constructed a homozygosity map of inbred AD cases extracted from an outbred population and explored ROH regions in whole-exome sequencing data (N = 1449). We detected a candidate marker, rs117458494, mapped in the SPON1 locus, which has been previously associated with amyloid metabolism. Here, we provide a research framework to look for recessive variants in AD using outbred populations. Our results showed that AD cases have enriched homozygosity, suggesting that recessive effects may explain a proportion of AD heritability.

Hardy Weinberg Equilibrium Disturbances in Case-Control Studies Lead to Non-Conclusive Results.

Recently, it has been proposed the association of a common deletion affecting toll-like receptor 2 promoter (-196 to -177) to type 2 diabetes mellitus risk. However, genotyping results show a significant deviation from the Hardy- Weinberg Equilibrium (HWE). The law of Hardy-Weinberg shows that for an autosomal biallelic marker with allele frequencies fA=p and fa=q, the proportion of subjects with genotypes AA, Aa, and aa should follow the following: fAA=p2, fAa=2pq, and faa=q2. Departure from HWE or Hardy-Weinberg Disequilibrium (HWD) in a human control population can be caused by natural factors such as selective pressure against a certain genotype. However their prevalence is scarce and magnitude of effect over the HWE are small. Other factors such as inbreeding caused by consanguinity, population stratification, or technical problems in genotyping are more usual. Nevertheless, if the control population follows a perfect HWE, the presence HWD among patients might be explained by the genetic association and evidencing a real link between the locus and the trait under study. However, HWD affecting both cases and controls, such as the one reported might be explained by one of the aforementioned issues.

White matter lesions and temporal atrophy are associated with cognitive and neuropsychiatric symptoms in patients with hypertension and Alzheimer's disease.

OBJECTIVES: An increasing evidence suggests hypertension (HTN) could be linked to cognitive impairment and incident Alzheimer's disease (AD). The precise mechanisms linking HTN and AD are not well-known. The aim of this study was to assess the putative association between HTN and AD. METHODS: We assessed in patients with AD associations between HTN and demographic and clinical data, vascular risk factors, treatments, APOE genotypes, brain white matter hyperintensities (WMH), and medial temporal atrophy (MTA) in multivariate analysis of covariance. RESULTS: We studied 92 patients with AD (mean ± SD age: 72.12 ± 6.91; women: 66.30%). Patients with HTN had significantly worse cognitive and functional status and higher frequency and severity of neuropsychiatric symptoms (P = .010). Magnetic resonance imaging analyzes showed significant increases in WMH (P = .018) and in MTA (P = .012) in patients with AD with HTN compared with those without HTN. CONCLUSIONS: Neuroimaging burden (MTA and higher degree of severity of WMH) among patients with AD and HTN are associated with the impaired cognitive function and neuropsychiatric symptoms.

Toll-Like Receptor 2 Promoter -196 to -174 Deletion Affects CD4 Levels Along Human Immunodeficiency Virus Infection Progression.

Toll-like receptor 2 (TLR2) plays a key role in innate immune response recognizing molecular patterns expressed by pathogens. rs111200466 is a TLR2 promoter insertion/deletion polymorphism with contradictory data about its role in human immunodeficiency virus type 1 (HIV-1) infection. We analyzed rs111200466 in HIV-1 disease progression and showed a correlation with a faster progression to the CD4+ < 200 cells/µL outcome for deletion allele carriers (Cox regression analysis: hazard ratio, 2.4 [95% confidence interval, 1.4-4]; P = .001). When naive patients with CD4+ < 200 cells/µL started antiretroviral treatment, rs111200466-deletion carriers showed a trend toward a slower, recovery rate (time required to reach CD4+ > 350 cells/µL; Cox P = .36). Our data suggest rs111200466 as a prognosis factor for HIV-1 disease progression.

Monoamino oxidase alleles correlate with the presence of essential hypertension among hypogonadic patients.

BACKGROUND: Monoamine oxidase (MAO) activity has been traditionally implicated in blood pressure through its effects on biogenic amine levels such as catecholamines, serotonin, and dopamine. Nowadays, this role is considered relegated to side-effects such as orthostatic hypotension and/or hypertensive crisis derived from MAO-inhibitory treatments in patients with psychiatric disease. METHODS: In the present work we have found an association between a polymorphic variant of MAOB gene and arterial hypertension in obese hypogonadic patients. The study cases comprised a series of 219 nondiabetic males with a body mass index ≥30 kg/m2 and aged <45 years. Hypogonadism was defined as subnormal testosterone concentrations, when free testosterone values ranged <65 pg/ml. RESULTS: MAOB rs3027452-A allele carriers were significantly over-represented among hypertensive (HT) patients (25.49%) in comparison to either the non-HT patients (10%, OR = 3.079 CI95 [1.364-6.952], p = .005, Chi-square test) and the control population series of nonobese nor hypogonadic males (also 10%, p = .003 Chi-square test). Upon adjusted, an independent association was shown with the hypogonadic group with hypertension when compared with nonhypertensive hypogonadics (Beta = 3.653, p = .005). When quantitative analysis was performed, hypertensive patients harboring rs3027452-A allele showed higher systolic blood pressure values (p = .038, Mann-Whitney U-test) as well as an increased Systolic-Diastolic range despite following HT treatment (∆mmHg 54 vs. 48 for rs3027452-A and rs3027452-G respectively, p-value .019, Mann-Whitney U-test). Previous studies on MAOB revealed that rs3027452-A allele has been correlated to a lower activity of the enzyme, what gives a functional evidence over our observation. CONCLUSION: If this result could be extrapolated to other hypertensive patient groups, it would implicate a review of the markers and therapeutic targets on human hypertension.

Genome-wide association analysis of dementia and its clinical endophenotypes reveal novel loci associated with Alzheimer's disease and three causality networks: The GR@ACE project.

INTRODUCTION: Large variability among Alzheimer's disease (AD) cases might impact genetic discoveries and complicate dissection of underlying biological pathways. METHODS: Genome Research at Fundacio ACE (GR@ACE) is a genome-wide study of dementia and its clinical endophenotypes, defined based on AD's clinical certainty and vascular burden. We assessed the impact of known AD loci across endophenotypes to generate loci categories. We incorporated gene coexpression data and conducted pathway analysis per category. Finally, to evaluate the effect of heterogeneity in genetic studies, GR@ACE series were meta-analyzed with additional genome-wide association study data sets. RESULTS: We classified known AD loci into three categories, which might reflect the disease clinical heterogeneity. Vascular processes were only detected as a causal mechanism in probable AD. The meta-analysis strategy revealed the ANKRD31-rs4704171 and NDUFAF6-rs10098778 and confirmed SCIMP-rs7225151 and CD33-rs3865444. DISCUSSION: The regulation of vasculature is a prominent causal component of probable AD. GR@ACE meta-analysis revealed novel AD genetic signals, strongly driven by the presence of clinical heterogeneity in the AD series.

Effects of SHBG rs1799941 Polymorphism on Free Testosterone Levels and Hypogonadism Risk in Young Non-Diabetic Obese Males.

INTRODUCTION: Obesity has been associated with increased risk of presenting hypogonadism. Free testosterone (FT) is the fraction of testosterone that carries out the biological function of testosterone, and is determined from total testosterone (TT) and sex-hormone binding globulin (SHBG) levels. We aimed to study the SHBG polymorphism rs1799941 in a cohort of young non-diabetic obese males to unravel the possible implication of this polymorphism in obesity-related hypogonadism. METHODOLOGY: 212 young (<45 years) non-diabetic obese (BMI ≥ 30 kg/m2) males participated in this study. Subjects were classified according to TT and FT levels in: Eugonadal (n = 55, TT > 3.5 ng/mL and FT ≥ 70 pg/mL; EuG), normal FT hypogonadism (n = 40, TT < 3.5 and FT ≥ 70 pg/mL; normal FT HG) and hypogonadism (n = 117, TT < 3.5 ng/mL and TL < 70 pg/mL; HG). The SHBG rs1799941 polymorphism (GG/GA/AA) was analyzed using the Taqman Open Array (Applied biosystem). RESULTS: The rs1799941 frequencies were different among the groups. Higher proportion of the allele (A) was found in HG, compared to EuG and normal FT HG. Among the genotypes, the rare homozygous (AA) were found in the normal FT HG group and higher levels of serum SHBG and lower of FT were observed. The presence of the allele A was related (according to lineal regression models) to an increased of SHBG levels ((GA) ß = 3.28; (AA) ß = 12.45) and a decreased of FT levels ((GA) ß = -9.19; (AA) ß = -18.52). The presence of the allele (A) increased the risk of presenting HG compared to normal FT HG (OR = 2.54). CONCLUSIONS: The rs1799941 of the SHBG gene can partially determine the presence of obesity-related hypogonadism in young non-diabetic males and whether these subjects have normal FT HG.

Comprehensive Toxicity Assessment of PEGylated Magnetic Nanoparticles for in vivo applications.

Magnetic nanoparticles (MNPs) represent one of the greatest promises for the development of a new generation of diagnostic agents for magnetic resonance imaging, with improved specificity and safety. Indeed, during the last decade the number of studies published in this field has grown exponentially. However, the clinical translation achieved so far has been very limited. This situation is likely related to the fact that most studies are focused on the in vitro characterization of these new nanomaterials, and very few provide an exhaustive in vivo characterization, where key aspects, such as pharmacokinetics, bioavailability, and, most importantly, toxicity, are properly evaluated. In this work, we propose a protocol for the comprehensive assessment of the toxicity of MNPs, based on the use of zebrafish embryos as an intermediate screening step between cell culture assays and studies in rodents. MNPs with different cores, ferrite and manganese ferrite oxide, and sizes between 3 and 20 nm, were evaluated. Cell viability at a concentration of 50 µg/mL of PEGylated MNPs was above 90 % in all cases. However, the exposure of zebrafish embryos to manganese based MNPs at concentrations above 100 µg/mL showed a low survival rate (<50 %). In contrast, no mortality (survival rate ∼100 %) and normal hatching rate were obtained for the iron oxide MNPs. Based on these results, together with the physicochemical and magnetic properties (r2 = 153.6 mM-1·s-1), the PEGylated 20 nm cubic shape iron oxide MNPs were selected and tested in mice, showing very good MRI contrast and, as expected, absence of toxicity.