Characterization of the SLC6A3 Promoter, A Gene Relevant to Substance Use Disorder
Open Access
- Author:
- Cardone, Katie
- Area of Honors:
- Biobehavioral Health
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- David John Vandenbergh, Thesis Supervisor
Helen Marie Kamens, Thesis Honors Advisor - Keywords:
- Genetics
Substance Use Disorder
Tandem Repeats
VNTR
STR
G-Quadruplexes - Abstract:
- Substance use disorder is a serious health issue. In 2020, 40.3 million Americans had a substance use disorder (Substance Abuse and Mental Health Services Administration, 2021) and 92,000 individuals died of overdose (Ahmad et al., 2021). Genetics play a role in substance use disorder (Goldman et al., 2005) and one gene, the human dopamine transporter gene or Solute Carrier, Family 3, Subfamily A, member 3 (SLC6A3), has been associated with substance use disorders (Reith et al., 2021). A few Variable Number of Tandem Repeats (VNTRs) loci have been identified in and around this gene that are suggested to differentially affect gene expression by acting through the gene’s promoter (Reith et al., 2021); however, little attention has been paid to shorter repeats (short tandem repeats or STRs). Given the role a gene’s promoter plays in gene expression (Tirosh et al., 2009), the first goal of this study was to identify whether an STR within the SLC6A3 promoter is variable. This STR has previously been identified in the Repeat Masker Track of the Genome Browser (accessed from http://genome.ucsc.edu). Although the SLC6A3 promoter has been studied, studies assessing variability of this STR or association studies with disease phenotypes were not described in the literature, so two methods were used to test for genetic variation in the STR. The first utilized polymerase chain reaction (PCR) and gel electrophoresis, which showed that the number of repeats does not vary between individuals. In the second method, 62 publicly-available-long-read genome sequences were aligned. Analysis confirmed that there was no variation in the number of repeats between alleles, but one single nucleotide polymorphism (SNP) and two novel insertions/deletions (in/dels) were identified. Recognition that this region was GC rich led to a second hypothesis that G-Quadruplexes (G4s) form in this STR (Kolesnikova and Curtis, 2019). Thus, the second goal of the project was to identify whether G-Quadruplexes formed, which was tested with an online prediction software and by circular dichroism (CD) spectroscopy. The CD Spectra showed that G4s form in this STR. These findings may have implications for gene expression because transcription factors bind to G4s (Stevens et al., 2017). Additionally, some variants identified are within transcription factor binding sites, further supporting a role for genetic variation in the promoter as relevant to regulation of expression of SLC6A3. In summary, this work increased understanding of genetic variation and secondary structure formation within the SLC6A3 promoter, but further research is needed to determine any effects on gene expression or substance use disorder development.