Genetic and functional characterization of candidate genes for complex psychiatric diseases using next-generation sequencing and cellular uptake assays

Complex phenotypes are the result of a complex interplay between genes and environmental factors. Extensive linkage, candidate and genome-wide association studies (GWASs) have been carried out to unravel genetic risk variants for human diseases. The identification of genes, involved in the pathomechanism of a disease, might be beneficial for its diagnosis, treatment and prognosis. While GWASs allowed the identification of a large number of common variants robustly associated with common complex diseases, the heritability, which can be explained by these variants, is small. The discrepancy between the estimated heritability from twin, family and adoption studies and the heritability obtained from GWAS was termed “missing heritability” and led to the investigation of additional factors that might also contribute to disease susceptibility, including gene-environment interactions, gene-gene interactions, structural variants and rare variants. In this thesis, the role of less common and rare variants in susceptibility to common complex diseases was investigated. In order to accomplish this, a candidate gene for panic disorder (PD) and a possible risk gene for major depressive disorder (MDD) were screened for the presence of common and rare variants using next-generation sequencing in a pooled approach. In a previously published GWAS, a haplotype containing two common intronic variants in the transmembrane protein 132D (TMEM132D) gene was associated with PD. Another GWAS identified solute carrier family 6 member 15 (SLC6A15), which encodes an amino acid transporter, as a risk gene for MDD. A common intergenic variant about 600 kilobase downstream of this gene was shown to decrease SLC6A15 gene expression in lymphoblastoid cell lines and hippocampus. Susceptibility genes for complex diseases, identified in GWAS, are promising candidates for the search of rare variants as genes harbouring common variants are likely to contain also rare variants. Pooled targeted re-sequencing of the exonic regions of TMEM132D in 300 anxiety disorder patients, mostly suffering from PD (84.7%), and 300 healthy controls allowed the detection of 371 genetic variants. Of these variants, 24.0% were common (minor allele frequency (MAF) > 5.0%), whereas the vast majority was less common (MAF 1.0 – 5.0%) to rare. 247 variants had not been reported before, including 12 novel non-synonymous variants leading to an amino acid exchange in the protein. While common variants associated with PD were not identified, an overrepresentation of non-synonymous variants and variants with predicted changes on splicing in healthy controls compared to PD patients was observed. These putatively functional relevant variants were distributed along a broad MAF spectrum, ranging from 0.17 to 30.0%. In addition, a higher rate of private non-synonymous variants, which were only present in either cases or controls in this study, but not in over 7,500 individuals with different ethnic backgrounds from other publicly available re-sequencing datasets, in patients compared to controls was seen. Combined with the data from the previous GWAS study in which the association with PD was carried by common variants, this pooled re-sequencing study suggests that not only common or rare variants alone, but a combination of both contributes to the development of anxiety-related phenotypes. Re-sequencing the whole SLC6A15 locus in 400 MDD patients and 400 healthy controls, 405 genetic variants were identified, including twelve non-synonymous variants. Only 15.0% of the detected variants were common. While none of the non-synonymous variants was significantly associated with MDD, two rare non-synonymous variants were identified to influence protein function. In contrast to the TMEM132D protein whose molecular function has still to be discovered, SLC6A15 is known to transport neutral amino acids into predominantly neuronal cells. The cellular uptake of neutral amino acids such as proline is thus a measurable property that associates with function. The uptake experiments identified two rare variants to be associated with a significant increase in proline uptake in HEK cells. This result suggests that rare variants in SLC6A15 might influence the biochemical function of its amino acid transporter and thus downstream neuronal function and possibly the risk for MDD and other stress-related psychiatric disorders. In addition, this study highlights that functional exploration of genetic variants might be promising to identify putatively disease-relevant variants as statistically significant associations for rare variants might only be achieved in extremely large samples

Phenocopy – A Strategy to Qualify Chemical Compounds during Hit-to-Lead and/or Lead Optimization

A phenocopy is defined as an environmentally induced phenotype of one individual which is identical to the genotype-determined phenotype of another individual. The phenocopy phenomenon has been translated to the drug discovery process as phenotypes produced by the treatment of biological systems with new chemical entities (NCE) may resemble environmentally induced phenotypic modifications. Various new chemical entities exerting inhibition of the kinase activity of Transforming Growth Factor β Receptor I (TGF-βR1) were qualified by high-throughput RNA expression profiling. This chemical genomics approach resulted in a precise time-dependent insight to the TGF-β biology and allowed furthermore a comprehensive analysis of each NCE's off-target effects. The evaluation of off-target effects by the phenocopy approach allows a more accurate and integrated view on optimized compounds, supplementing classical biological evaluation parameters such as potency and selectivity. It has therefore the potential to become a novel method for ranking compounds during various drug discovery phases

vipR: variant identification in pooled DNA using R

Motivation: High-throughput-sequencing (HTS) technologies are the method of choice for screening the human genome for rare sequence variants causing susceptibility to complex diseases. Unfortunately, preparation of samples for a large number of individuals is still very cost- and labor intensive. Thus, recently, screens for rare sequence variants were carried out in samples of pooled DNA, in which equimolar amounts of DNA from multiple individuals are mixed prior to sequencing with HTS. The resulting sequence data, however, poses a bioinformatics challenge: the discrimination of sequencing errors from real sequence variants present at a low frequency in the DNA pool

Comparison of normalization methods for Illumina BeadChip HumanHT-12 v3

Abstract Background Normalization of microarrays is a standard practice to account for and minimize effects which are not due to the controlled factors in an experiment. There is an overwhelming number of different methods that can be applied, none of which is ideally suited for all experimental designs. Thus, it is important to identify a normalization method appropriate for the experimental setup under consideration that is neither too negligent nor too stringent. Major aim is to derive optimal results from the underlying experiment. Comparisons of different normalization methods have already been conducted, none of which, to our knowledge, comparing more than a handful of methods. Results In the present study, 25 different ways of pre-processing Illumina Sentrix BeadChip array data are compared. Among others, methods provided by the BeadStudio software are taken into account. Looking at different statistical measures, we point out the ideal versus the actual observations. Additionally, we compare qRT-PCR measurements of transcripts from different ranges of expression intensities to the respective normalized values of the microarray data. Taking together all different kinds of measures, the ideal method for our dataset is identified. Conclusions Pre-processing of microarray gene expression experiments has been shown to influence further downstream analysis to a great extent and thus has to be carefully chosen based on the design of the experiment. This study provides a recommendation for deciding which normalization method is best suited for a particular experimental setup.</p

Rare variants in TMEM132D in a case-control sample for panic disorder

Genome-wide association studies have identified common variants associated with common diseases. Most variants, however, explain only a small proportion of the estimated heritability, suggesting that rare variants might contribute to a larger extent to common diseases than assumed to date. Here, we use next-generation sequencing to test whether such variants contribute to the risk for anxiety disorders by re-sequencing 40 kb including all exons of the TMEM132D locus which we have previously shown to be associated with panic disorder and anxiety severity measures. DNA from 300 patients suffering from anxiety disorders, mostly panic disorder (84.7%), and 300 healthy controls was screened for the presence of genetic variants using next-generation re-sequencing in a pooled approach. Results were verified by individual re-genotyping. We identified 371 variants of which 247 had not been reported before, including 15 novel non-synonymous variants. The majority, 76% of these variants had a minor allele frequency less than 5%. While we did not identify additional common variants in TMEM132D associated with panic disorders, we observed an overrepresentation of presumably functional coding variants in healthy controls as compared to cases as well as a higher rate of private coding variants in cases, with one non-synonymous coding variant present in four patients but not in any of the matched controls nor in over 5,500 individuals of different ethnic origins from publicly available re-sequencing datasets. Our data suggest that not only common but also putatively functional and/or rare variants within TMEM132D might contribute to the risk to develop anxiety disorders

The 5-HTTLPR polymorphism modulates the influence on environmental stressors on peripartum depression symptoms

Background: Maternal depression during the peripartum period has an incidence of about 13%. Individuals with specific genetic predispositions are more vulnerable to stressful life events suggesting that exploration of gene-environmental pathways might facilitate the identification of risk factors for peripartum depression. The aim of this study was to evaluate the influence of stressful life events in combination with the serotonin transporter gene 5-HTTLPR polymorphism on peripartum depressive symptoms. Methods: In a non-psychiatric cohort of 419 Caucasians, the severity of depression was assessed prospectively during pregnancy (3rd trimester) and the postpartum period (2-3 days and 6-8 months) using the Edinburgh Postnatal Depression Scale. Satisfaction with the partner and exposure to negative life events were evaluated using self-report questionnaires and the genotype of the 5-HTTLPR was assessed. Repeated measures generalized linear models were used to investigate the gene-environment interaction on depressive symptoms across late pregnancy and the postpartum period. Results: The 5-HTTLPR S-allele carrier status predicted late postpartum depressive symptom severity only in the presence of negative life events. This interaction was not observed for depressive symptoms during the 3rd trimester or the early postpartum. In addition, S-allele carrier status increased the negative effects of dissatisfaction with the current partner on depressive symptoms in the late postpartum period. Conclusions: In this non-psychiatric cohort, the 5-HTTLPR interacts with both lifetime and current stressors to influence depressive symptoms in the late post partum period. These findings could have clinical implications by allowing identification of women at higher risk for developing postpartum depressive symptoms

Functional Coding Variants in <i>SLC6A15</i>, a Possible Risk Gene for Major Depression

<div><p>SLC6A15 is a neuron-specific neutral amino acid transporter that belongs to the solute carrier 6 gene family. This gene family is responsible for presynaptic re-uptake of the majority of neurotransmitters. Convergent data from human studies, animal models and pharmacological investigations suggest a possible role of SLC6A15 in major depressive disorder. In this work, we explored potential functional variants in this gene that could influence the activity of the amino acid transporter and thus downstream neuronal function and possibly the risk for stress-related psychiatric disorders. DNA from 400 depressed patients and 400 controls was screened for genetic variants using a pooled targeted re-sequencing approach. Results were verified by individual re-genotyping and validated non-synonymous coding variants were tested in an independent sample (N = 1934). Nine variants altering the amino acid sequence were then assessed for their functional effects by measuring SLC6A15 transporter activity in a cellular uptake assay. In total, we identified 405 genetic variants, including twelve non-synonymous variants. While none of the non-synonymous coding variants showed significant differences in case-control associations, two rare non-synonymous variants were associated with a significantly increased maximal ³H proline uptake as compared to the wildtype sequence. Our data suggest that genetic variants in the <i>SLC6A15</i> locus change the activity of the amino acid transporter and might thus influence its neuronal function and the risk for stress-related psychiatric disorders. As statistically significant association for rare variants might only be achieved in extremely large samples (N >70,000) functional exploration may shed light on putatively disease-relevant variants.</p></div

Genetic Variants in the Genes of the Stress Hormone Signalling Pathway and Depressive Symptoms during and after Pregnancy

Purpose. The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in genes of the stress hormone signaling pathway, specifically FKBP5, NR3C1, and CRHR1, are associated with depressive symptoms during and after pregnancy. Methods. The Franconian Maternal Health Evaluation Study (FRAMES) recruited healthy pregnant women prospectively for the assessment of maternal and fetal health including the assessment of depressiveness. The German version of the 10-item Edinburgh Postnatal Depression Scale (EPDS) was completed at three time points in this prospective cohort study. Visit 1 was at study entry in the third trimester of the pregnancy, visit 2 was shortly after birth, and visit 3 was 6-8 months after birth. Germline DNA was collected from 361 pregnant women. Nine SNPs in the above mentioned genes were genotyped. After construction of haplotypes for each gene, a multifactorial linear mixed model was performed to analyse the depression values over time. Results. EPDS values were within expected ranges and comparable to previously published studies. Neither did the depression scores differ for comparisons among haplotypes at fixed time points nor did the change over time differ among haplotypes for the examined genes. No haplotype showed significant associations with depressive symptoms severity during pregnancy or the postpartum period. Conclusion. The analysed candidate haplotypes in FKBP5, NR3C1, and CRHR1 did not show an association with depression scores as assessed by EPDS in this cohort of healthy unselected pregnant women

Gender-specific association of variants in the akr1c1 gene with dimensional anxiety in patients with panic disorder: additional evidence for the importance of neurosteroids in anxiety?

BackgroundNeurosteroids are synthesized both in brain and peripheral steroidogenic tissue from cholesterol or steroidal precursors. Neurosteroids have been shown to be implicated in neural proliferation, differentiation, and activity. Preclinical and clinical studies also suggest a modulatory role of neurosteroids in anxiety-related phenotypes. However, little is known about the contribution of genetic variants in genes relevant for the neurosteroidogenesis to anxiety disorders. MethodsWe performed an association analysis of single nucleotide polymorphisms (SNPs) in five genes related to the neurosteroidal pathway with emphasis on progesterone and allopregnanolone biosynthesis (steroid-5-alpha-reductase 1A (SRD5A1), aldo-keto reductase family 1 C1-C3 (AKR1C1-AKR1C3) and translocator protein 18 kDA (TSPO) with panic disorder (PD) and dimensional anxiety in two German PD samples (cases N = 522, controls N = 1,115). ResultsCase-control analysis for PD and SNPs in the five selected genes was negative in the combined sample. However, we detected a significant association of anticipatory anxiety with two intronic SNPs (rs3930965, rs41314625) located in the gene AKR1C1 surviving correction for multiple testing in PD patients. Stratification analysis for gender revealed a female-specific effect of the associations of both SNPs. ConclusionsThese results suggest a modulatory effect of AKR1C1 activity on anxiety levels, most likely through changes in progesterone and allopregnanolone levels within and outside the brain. In summary, this is the first evidence for the gender-specific implication of the AKR1C1 gene in the expression of anticipatory anxiety in PD. Further analyses to unravel the functional role of the SNPs detected here and replication analyses are needed to validate our results