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) \ensuremath> 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

Respecting Legislators and Rejecting Baselines: Rebalancing Casey

Part I of this Note describes the background cases leading to the Supreme Court’s decision in Casey and the resulting undue burden standard. This Part also explains the limited circumstances in which the undue burden standard gives more definitive guidelines for judicial decisionmaking. Part II works through several federal district and appellate court cases to identify some of the underlying baseline presumptions and normative value judgments influencing judicial decisions in this area of the law. These baselines are often dispositive in determining whether a restriction on abortion is due or undue, cutting against the goodwill attempts by legislatures to make abortion as safe as possible while adhering to the Supreme Court’s constitutional protection of the procedure. Importantly, this Note does not impute upon judges any bad faith or malfeasance in relation to their adjudications in this area, but rather seeks to emphasize the inherent vagueness of the undue burden standard. Part III analyzes the impropriety of judicially created baselines and calls judges to account more clearly for their values on these matters when using the undue burden standard. As a threshold matter, this Note does not support the undue burden standard as stated in Casey. While the use of a balancing test in important and controversial constitutional matters may be appropriate at times, the underlying premise of the Casey undue burden standard is deeply flawed. While at once stating that women should be free to decide when life begins, the Court demands that for all states an unborn child is only a potential life, rather than an actual life, and establishes the balancing test using those unwavering parameters. States should have the authority to decide that a fetus, as an unquestionably distinct member of the human species, is a person whose full value needs to be balanced against the liberty interests of his or her mother. Such a standard would both allow state lawmakers who value unborn human life to make laws to properly defend it, while giving other states the freedom to deny the personhood and value of the unborn or instead identify situations where liberty interests are deemed more valuable than life

Respecting Legislators and Rejecting Baselines: Rebalancing Casey

Part I of this Note describes the background cases leading to the Supreme Court’s decision in Casey and the resulting undue burden standard. This Part also explains the limited circumstances in which the undue burden standard gives more definitive guidelines for judicial decisionmaking. Part II works through several federal district and appellate court cases to identify some of the underlying baseline presumptions and normative value judgments influencing judicial decisions in this area of the law. These baselines are often dispositive in determining whether a restriction on abortion is due or undue, cutting against the goodwill attempts by legislatures to make abortion as safe as possible while adhering to the Supreme Court’s constitutional protection of the procedure. Importantly, this Note does not impute upon judges any bad faith or malfeasance in relation to their adjudications in this area, but rather seeks to emphasize the inherent vagueness of the undue burden standard. Part III analyzes the impropriety of judicially created baselines and calls judges to account more clearly for their values on these matters when using the undue burden standard. As a threshold matter, this Note does not support the undue burden standard as stated in Casey. While the use of a balancing test in important and controversial constitutional matters may be appropriate at times, the underlying premise of the Casey undue burden standard is deeply flawed. While at once stating that women should be free to decide when life begins, the Court demands that for all states an unborn child is only a potential life, rather than an actual life, and establishes the balancing test using those unwavering parameters. States should have the authority to decide that a fetus, as an unquestionably distinct member of the human species, is a person whose full value needs to be balanced against the liberty interests of his or her mother. Such a standard would both allow state lawmakers who value unborn human life to make laws to properly defend it, while giving other states the freedom to deny the personhood and value of the unborn or instead identify situations where liberty interests are deemed more valuable than life

Reasonable Minds May Differ: The Application of Miller and Graham to Consecutive Sentences for Juvenile Offenders in Missouri

Ledale Nathan was convicted of second-degree murder and a series of nonhomicide offenses stemming from a home invasion he committed at the age of sixteen. The St. Louis Circuit Court sentenced Nathan at a time when Eighth Amendment jurisprudence regarding juvenile sentencing was in flux. The United States Supreme Court decided two cases that restricted the way courts sentence juvenile offenders: Graham v. Florida and Miller v. Alabama. Graham held that juvenile offenders who had not committed homicide offenses could not be sentenced to life without parole (“LWOP”), while Miller held that LWOP could not be a mandatory sentence for juvenile homicide offenders. However, Graham and Miller left two main questions unresolved. First, does Graham bar consecutive sentences for multiple nonhomicide offenses that effectively function as LWOP? Second, when a juvenile is convicted of homicide and nonhomicide offenses, does Miller require courts to consider mitigating evidence when imposing a sentence for the homicide offense alone, or must courts consider such evidence when imposing aggregate sentences for both the homicide and nonhomicide offenses

Search for associations containing young stars (SACY). V. Is multiplicity universal? Tight multiple systems

Context: Dynamically undisrupted, young populations of stars are crucial to study the role of multiplicity in relation to star formation. Loose nearby associations provide us with a great sample of close ($<$150 pc) Pre-Main Sequence (PMS) stars across the very important age range ($\approx$5-70 Myr) to conduct such research. Aims: We characterize the short period multiplicity fraction of the SACY (Search for Associations Containing Young stars) accounting for any identifiable bias in our techniques and present the role of multiplicity fractions of the SACY sample in the context of star formation. Methods: Using the cross-correlation technique we identified double-lined spectroscopic systems (SB2), in addition to this we computed Radial Velocity (RV) values for our subsample of SACY targets using several epochs of FEROS and UVES data. These values were used to revise the membership of each association then combined with archival data to determine significant RV variations across different data epochs characteristic of multiplicity; single-lined multiple systems (SB1). Results: We identified 7 new multiple systems (SB1s: 5, SB2s: 2). We find no significant difference between the short period multiplicity fraction ($F_\mathrm{m}$) of the SACY sample and that of nearby star forming regions ($\approx$1-2 Myr) and the field ($F_\mathrm{m}\leq$10%) both as a function of age and as a function of primary mass, $M_1$, in the ranges $P$ [1:200 day] and $M_2$ [0.08 $M_{\odot}$-$M_1$]. Conclusions: Our results are consistent with the picture of universal star formation, when compared to the field and nearby star forming regions (SFRs). We comment on the implications of the relationship between increasing multiplicity fraction with primary mass, within the close companion range, in relation to star formation.Comment: 14 pages, 18 figures, published, A&A http://dx.doi.org/10.1051/0004-6361/20142385