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