Evaluation of methods for meta-analysis of genetic linkage studies for complex diseases and application to genome scans for asthma and adult height

Linkage genome scans for genetically complex diseases have low power with the sample sizes that were often used in the past, and hence meta-analysis of several scans for the same disease might be a promising approach. Appropriate data are now becoming accessible as many groups worldwide investigate common diseases. The aim of this thesis is to extend and evaluate statistical methodology for meta-analysis. In addition, two meta-analyses of linkage genome scans for the complex phenotypes asthma and adult stature are performed and discussed. In the first part of this thesis, an overview of available statistical methods and current applications is given. A new meta-analysis method is introduced which is based on a weighted combination of non-parametric linkage scores. Its relationship to traditional fixed effects meta-analysis of combining parameter estimates from different studies weighted by the inverse of their respective variances is described. Recombination and low informativity of markers lead to a reduction of the effective sample size in multipoint linkage analysis. A locus specific weighting of individual studies with this effective sample size is therefore proposed. In a simulation study, the power of different methods to combine multipoint linkage scores, namely Fisher’s p-value combination (Fisher 1932), the truncated product method (Zaykin et al. 2002, a variant of Fisher's method), the Genome Search Meta-Analysis (GSMA, Wise et al. 1999) method and the proposed weighting methods were compared. In particular, the effects of different genetic marker sets and sample sizes between genome scans were investigated. The weighting methods explicitly take those differences into account and have higher power in the simulated scenarios than the other methods. The proposed meta-analysis method was applied to four linkage genome scans for the phenotype asthma and five studies of a candidate genetic region. Multipoint nonparametric linkage analysis is performed and different weighting schemes are used to combine the score statistics of individual studies to an overall statistic. For comparison, the GSMA method is also applied to the same data sets. For meta-analysis of linkage studies, a common map of genetic markers is necessary to align results obtained in different studies with different markers. In this meta-analysis, the effects of map uncertainties were evaluated. The latest versions of available combined physical and linkage maps are very precise and the small potential map errors that are left do not have relevant impact. This meta-analysis of nine asthma linkage studies does not identify significant regions of genetic linkage to asthma. A still rather small size of the combined samples may be the reason for low power to identify susceptibility genes for the complex trait asthma. The statistical methods that can be applied for a meta-analysis of linkage studies depend crucially on the available data, especially any additional information besides the usually reported linkage statistics. For the meta-analysis of linkage genome scans for the highly heritable trait adult height, only LOD scores from variance components linkage analysis, which are measures of significance and not effect estimates, could be obtained. Thus, Fisher’s method and a weighted and unweighted variant of the inverse normal method were applied. Initially, a linkage genome scan for this quantitative trait was performed in the extended pedigrees of the Framingham Heart Study. A variance components linkage analysis in this sample unselected for height gave evidence for linkage in several regions. All markers showing a LOD score greater than 1 in this analysis correspond to previously reported linkage regions, including chromosome 6q with a maximum LOD score of 2.45 and chromosomes 9, 12, 14, 18 and 22. Following this observation, a meta-analysis of all previously published genome scans for adult stature was planned. Genome scan results of 17 separate samples reported in seven publications and comprising more than 14000 phenotyped and genotyped individuals could be obtained in sufficient detail to be included in the meta-analysis. The comparison of meta-analysis results with individual studies shows that only a formal meta-analysis can exactly quantify the combined evidence for linkage and is superior to an informal classification of results as replication or non-replication. Significant linkage of stature is observed on chromosomes 6, 7, 9 and 12 (LOD scores >4) and suggestive linkage with LOD scores >2 is obtained in six additional genetic regions. This is well compatible with the concept of height as a mostly polygenic trait for which also some major genes exist. Candidate genes in the linkage regions are discussed

Genome scan for body mass index and height in the Framingham Heart Study

BACKGROUND: Body mass index (BMI) and adult height are moderately and highly heritable traits, respectively. To investigate the genetic background of these quantitative phenotypes, we performed a linkage genome scan in the extended pedigrees of the Framingham Heart Study. Two variance-components approaches (SOLAR and MERLIN-VC) and one regression method (MERLIN-REGRESS) were applied to the data. RESULTS: Evidence for linkage to BMI was found on chromosomes 16 and 6 with maximum LOD scores of 3.2 and 2.7, respectively. For height, all markers showing a LOD score greater than 1 in our analysis correspond to previously reported linkage regions, including chromosome 6q with a maximum LOD score of 2.45 and chromosomes 9, 12, 14, 18, and 22. Regarding the analysis, the three applied methods gave very similar results in this unselected sample with approximately normally distributed traits. CONCLUSION: Our analysis resulted in the successful identification of linked regions. In particular, we consider the regions on chromosomes 6 and 16 for BMI and the regions on chromosomes 6, 9, and 12 for stature interesting for fine mapping and candidate gene studies

Motivation to Change in the Course of a Pilot Study of a Step-Down Treatment Approach of Inpatient and Anorexia Nervosa-Specific Home Treatment and Its Effects on Treatment Outcome

Introduction: Anorexia nervosa (AN) is a serious mental disorder that typically manifests in adolescence. Motivation to change is an important predictor for treatment outcome in adolescent AN, even though its development over the often long therapeutic process, with transitions between treatment settings, has not yet been studied. In this pilot study, the course of motivation to change and its effect on treatment outcome were investigated over the course of a step-down treatment approach during a 12-month observation period. Methods: Twenty-one adolescents admitted to inpatient treatment because of AN received multidisciplinary home treatment (HoT) with several weekly visits after short inpatient stabilization. Eating disorder (ED-)specific cognitive [Eating Disorder Inventory 2 (EDI-2) subscales] and physical [% expected body weight (%EBW)] illness severity and motivation to change [Anorexia Nervosa Stages of Change Questionnaire (ANSOCQ)] were assessed at the time of admission, discharge from hospital, at the end of HoT, and at a 12-month follow-up. Changes in motivation over time and its relationship with treatment outcome were investigated. Results: Mean motivation to change improved significantly over the course of treatment from the contemplation stage [2nd stage, mean ANSOCQ sum score 47.26 (SD 17.60)] at admission to the action stage [4th stage, mean ANSOCQ sum score 77.64 (SD 18.97)] at the end of HoT (p < 0.001) and remained stable during the follow-up period. At each assessment, higher motivation to change was significantly correlated with lower ED-specific cognitive illness severity (Spearman ρs: -0.53 to -0.77, all p < 0.05). Only pretreatment motivation to change significantly predicted ED-specific cognitive illness severity after the first inpatient treatment phase when taking prior illness severity into account. Conclusions: Motivation to change is an important aspect of treatment success in adolescent AN, especially in the early phase of treatment. In addition, home treatment contributed significantly to a higher motivation. Further longitudinal research into how motivation to change in adolescent patients with AN is related to outcome in this often severe and enduring disease and into targeted therapeutic strategies and interventions that reliably enhance the motivation to change in adolescent patients with AN seems promising

The Impact of Starvation on the Microbiome and Gut-Brain Interaction in Anorexia Nervosa

Interactions between the gut microbiome and the brain are of increasing interest to both researchers and clinicians. Evidence is mounting on the causal role of an altered gut microbiome in inflammatory diseases such as arthritis, inflammatory bowel disease, obesity and diabetes, and psychiatric diseases like anxiety and depression. Mechanisms include altered energy harvest from food, hormonal changes, increased gut permeability, inflammation, immune response, and a direct influence on the brain and behavior. Anorexia nervosa (AN) is the third most common disease in adolescence and exacts a high burden on patients and caregivers. It often becomes chronic and has the highest mortality of all psychiatric diseases. As AN is characterized by nutritional restrictions, weight loss, and severe behavioral symptoms including weight phobia, comorbid anxiety and depression, accompanied by endocrine alterations, increased inflammation, and immune response, exploring the role of the gut microbiome is crucial. Here, we present an overview of the potential mechanisms of interaction between the gut microbiome, the host and particularly the brain in AN and summarize the initial findings of microbiome research on AN. We conclude by identifying future research directions and potential therapeutic approaches, including nutritional interventions, probiotics, prebiotics and food supplements, that could become important additions to current AN therapy

Gut microbiota alteration in adolescent anorexia nervosa does not normalize with short-term weight restoration

Objective Gut microbiota are linked to metabolic function, body weight regulation, and brain and behavioral changes. Alteration of gut microbiota is repeatedly demonstrated in adults with anorexia nervosa (AN) and transplantation of stool from adult patients with AN reduces weight gain, food consumption and food efficiency in germ-free mice. No similar data are available for adolescents, who might differ from adults due to their shorter duration of illness. Method Nineteen female adolescent patients with AN at admission and after short-term weight recovery were included in a longitudinal study and compared to 20 healthy controls (HC). DNA was extracted from stool samples and subjected to 16S rRNA gene sequencing and analysis. Group comparisons, indicator genera and simper analysis were applied. Taxon abundances at admission was used to predict inpatient treatment duration. Results Alpha diversity is increased in patients with AN after short-term weight recovery, while beta diversity shows clear group differences with HC before and after weight gain. A reduction in Romboutsia and taxa belonging to Enterobacteriaceae at both timepoints and an increase in taxa belonging to Lachnospiraceae at discharge are most indicative of patients. Lachnospiraceae abundance at admission helped to predict shorter inpatient treatment duration. Discussion This pilot study provides first evidence of gut microbiota alterations in adolescent patients with AN that do not normalize with weight gain. If verified in larger studies, the predictive power of taxa belonging to Lachnospiraceae for clinical outcome could complement known predictors at admission, inform clinicians and serve as a target for nutritional interventions

Combined PARP and Dual Topoisomerase Inhibition Potentiates Genome Instability and Cell Death in Ovarian Cancer

Although ovarian cancer is a rare disease, it constitutes the fifth leading cause of cancer death among women. It is of major importance to develop new therapeutic strategies to improve survival. Combining P8-D6, a novel dual topoisomerase inhibitor with exceptional anti-tumoral properties in ovarian cancer and compounds in preclinical research, and olaparib, a PARP inhibitor targeting DNA damage repair, is a promising approach. P8-D6 induces DNA damage that can be repaired by base excision repair or homologous recombination in which PARP plays a major role. This study analyzed benefits of combining P8-D6 and olaparib treatment in 2D and 3D cultures with ovarian cancer cells. Measurement of viability, cytotoxicity and caspase activity were used to assess therapy efficacy and to calculate the combination index (CI). Further DNA damage was quantified using the biomarkers RAD51 and γH2A.X. The combinational treatment led to an increased caspase activity and reduced viability. CI values partially show synergisms in combinations at 100 nM and 500 nM P8-D6. More DNA damage accumulated, and spheroids lost their membrane integrity due to the combinational treatment. While maintaining the same therapy efficacy as single-drug therapy, doses of P8-D6 and olaparib can be reduced in combinational treatments. Synergisms can be seen in some tested combinations. In summary, the combination therapy indicates benefits and acts synergistic at 100 nM and 500 nM P8-D6

Improvement of renal function after transcatheter aortic valve replacement and its impact on survival

Background Chronic kidney disease as well as acute kidney injury are associated with adverse outcomes after transcatheter aortic valve replacement (TAVR). However, little is known about the prognostic implications of an improvement in renal function after TAVR. Methods Renal improvement (RI) was defined as a decrease in postprocedural creatinine in μmol/l of ≥1% compared to its preprocedural baseline value. A propensity score representing the likelihood of RI was calculated to define patient groups which were comparable regarding potential confounders (age, sex, BMI, NYHA classification, STS score, log. EuroSCORE, history of atrial fibrillation/atrial flutter, pulmonary disease, previous stroke, CRP, creatinine, hsTNT and NT-proBNP). The cohort was stratified into 5 quintiles according to this propensity score and the survival time after TAVR was compared within each subgroup. Results Patients in quintile 5 (n = 93) had the highest likelihood for RI. They were characterized by higher creatinine, lower eGFR, higher NYHA class, higher NT-proBNP, being mostly female and having shorter overall survival time. Within quintile 5, patients without RI had significantly shorter survival compared to patients with RI (p = 0.002, HR = 0.32, 95% CI = [0.15-0.69]). There was no survival time difference between patients with and without RI in the whole cohort (p = 0.12) and in quintiles 1 to 4 (all p > 0.16). Analyses of specific subgroups showed that among patients with NYHA class IV, those with RI also had a significant survival time benefit (p < 0.001, HR = 0.15; 95%-CI = [0.05-0.44]) compared to patients without RI. Conclusions We here describe a propensity score-derived specific subgroup of patients in which RI after TAVR correlated with a significant survival benefit

Lower serum levels of IL-1β and IL-6 cytokines in adolescents with anorexia nervosa and their association with gut microbiota in a longitudinal study

Introduction Anorexia nervosa (AN) is an often chronic and debilitating psychiatric disease whose etiology is not completely understood. Recently, a potential role of inflammation has emerged in other psychiatric diseases, such as depression, PTSD and schizophrenia. The first results in adults with AN seemed to confirm a low-grade proinflammatory state until recent studies presented more differential findings. Studying adolescents with a shorter illness duration and fewer confounding factors might help elucidate the role of inflammation in the underlying pathophysiology of AN; however, the few available studies in adolescents remain ambiguous, and no longitudinal data are available in this age range. Results TNF-α serum levels were significantly elevated in patients with AN at admission, while IL-1β and IL-6 levels were lower at admission and discharge than in HC. After treatment, we also found significantly elevated levels of IL-6 Rα compared to HC, while IL-15 did not show significant changes. Exploratory analyses revealed positive associations of cytokine and genus-level changes between admission and discharge for IL-1β (Bacteroides) and IL-15 (Romboutsia), and negative associations for IL-15 (Anaerostipes) and TNF-α (uncultured Lachnospiraceae). Conclusion We confirmed a previous finding of elevated levels of TNF-α also in adolescents with AN; however, the reduced IL-1β and IL-6 levels differed from the mostly increased levels found in adults. A mixed pro- and anti-inflammatory state appears to be present in adolescents, potentially due to their shorter illness duration. The gut microbiota, with its regulatory function on cytokine production, might play a role in mediating these inflammatory processes in AN and could offer targets for new therapeutic approaches

Long-Term Neuroanatomical Consequences of Childhood Maltreatment: Reduced Amygdala Inhibition by Medial Prefrontal Cortex

Similar to patients with Major depressive disorder (MDD), healthy subjects at risk for depression show hyperactivation of the amygdala as a response to negative emotional expressions. The medial prefrontal cortex is responsible for amygdala control. Analyzing a large cohort of healthy subjects, we aimed to delineate malfunction in amygdala regulation by the medial prefrontal cortex in subjects at increased risk for depression, i.e., with a family history of affective disorders or a personal history of childhood maltreatment. We included a total of 342 healthy subjects from the FOR2107 cohort (www.for2107.de). An emotional face-matching task was used to identify the medial prefrontal cortex and right amygdala. Dynamic Causal Modeling (DCM) was conducted and neural coupling parameters were obtained for healthy controls with and without particular risk factors for depression. We assigned a genetic risk if subjects had a first-degree relative with an affective disorder and an environmental risk if subjects experienced childhood maltreatment. We then compared amygdala inhibition during emotion processing between groups. Amygdala inhibition by the medial prefrontal cortex was present in subjects without those two risk factors, as indicated by negative model parameter estimates. Having a genetic risk (i.e., a family history) did not result in changes in amygdala inhibition compared to no risk subjects. In contrast, childhood maltreatment as environmental risk has led to a significant reduction of amygdala inhibition by the medial prefrontal cortex. We propose a mechanistic explanation for the amygdala hyperactivity in subjects with particular risk for depression, in particular childhood maltreatment, caused by a malfunctioned amygdala downregulation via the medial prefrontal cortex. As childhood maltreatment is a major environmental risk factor for depression, we emphasize the importance of this potential early biomarker