Analysis of Single Nucleotide Polymorphisms (SNPs) associated with Attention-Deficit/Hyperactivity Disorder (ADHD)




Jamie R. Listokin

Introduction

Attention-Deficit/Hyperactivity Disorder (ADHD) is characterized by symptoms of inattention, hyperactivity, and/or impulsivity (American Psychological Association, 2000).

Three types of presentations:
  • Predominantly hyperactive/impulsive presentation
  • Predominantly inattentive presentation
  • Combined presentation

ADHD children may suffer from deficits in overall self-control, leading to classic symptoms of hyperactivity and poor emotion regulation (Kofler, Rapport, Sarver, Raiker, Orban, Friedman & Kolomeyer, 2013).

Genetic Factors
  • Pooled results from 20 twin studies indicate the heritability of ADHD is 76% (Faraone et al., 2005).

  • Findings from longitudinal twin studies suggest that the continuity of ADHD symptoms from childhood to adolescence is associated with genetic influences (Kuntsi et al., 2005).

  • Genetic studies of quantitative ADHD symptom scores in children further support the idea that genetic factors may related to symptom severity (Anttila et al., 2018).

Previous GWAS Study

A previous genome-wide association study (GWAS) (Demontis et al., 2018) on 20,183 individuals with ADHD and 35,191 controls were collected from 12 European, North American, and Chinese cohorts.
  • In total, 304 genetic variants have been associated with ADHD. Of those, 12 variants surpassed the threshold for genome-wide significance (P < 5×10−8).
Two of the SNP variants occur in the SEMA6D and SORCS3 genes.
  • The SEMA6D gene was significantly associated with increased risk for ADHD and putamen volume (Hawi, 2018).

  • The SORCS3 gene has been to be a risk factor for both ADHD (Lesch, 2008). and bipolar disorder (Ollila, 2009).

Methods

The goal of the current study was to develop an assay for the presence for a SNP in selected genes.
  1. Primers were designed for:

    • rs281324 in the SEMA6D gene

    • rs11591402 in the SORCS3 gene

  2. A PCR reaction was performed.

  3. Products were visualized on 1% agarose gels, purified and sent out for sequencing.

  4. We sequenced the region including the SNPs.
This was a proof of principle; therefore, we did this on 10 de-identified participants.
Results

SEMA6D Results:

    Figure 1. Gel Electrophoresis Results of SEMA6D PCR
  • Five participants were homozygous for the T allele
    • "Normal” sequence
  • Three participants were homozygous for the C allele.
    • SNP associated with ADHD
  • One participant was heterozygous for the T and C alleles
    • "Normal" sequence and SNP associated with ADHD.
    Figure 2. Chromatogram of heterozygous rs281324 SNP

SORCS3 Results:

    Figure 3. Gel Electrophoresis Results of SORCS3 PCR

There were long stretches of repeat nucleotides in this PCR product.
  • As a result of using Taq, it is very difficult to interpret the sequencing.

    Figure 4. Chromatogram of SORCS3 results

Discussion

In the SEMA6D gene, we were successfully able to amplify the region where the SNP is. Normal and ADHD-associated SNPs were detected. The analysis sets the stage for a larger sample in a targeted population.
  • If this was to be conducted on a larger scale, we would do a more efficient analyses method, such as Taqman or Allelle Specific Analyses.

In the SORCS3 gene, we were unsuccessful in generating readable sequences of the region of the SNP.
  • More stringent sequencing methodology needs to be applied in order to sequence this region.

Refences
  1. American Psychological Association. (2000). Diagnostic and Statistical Manual of Mental Disorders (4th ed., Text Revision). Washington, DC.
  2. Anttila, V., Bulik-Sullivan, B., Finucane, H. K., Walters, R. K., Bras, J., Duncan, L., ... & Patsopoulos, N. A. (2018). Analysis of shared heritability in common disorders of the brain. Science, 360(6395), eaap8757.
  3. Faraone, S. V., Perlis, R. H., Doyle, A. E., Smoller, J. W., Goralnick, J. J., Holmgren, M. A., & Sklar, P. (2005). Molecular genetics of attention-deficit/hyperactivity disorder. Biological Psychiatry, 57(11), 1313-1323.
  4. Franke, B., Faraone, S. V., Asherson, P., Buitelaar, J., Bau, C. H. D., Ramos-Quiroga, J. A., ... & Lesch, K. P. (2012). The genetics of attention deficit/hyperactivity disorder in adults, a review. Molecular Psychiatry, 17(10), 960.
  5. Demontis, D., Walters, R. K., Martin, J., Mattheisen, M., Als, T. D., Agerbo, E., ... & Cerrato, F. (2019). Discovery of the first genome-wide significant risk loci for attention deficit/hyperactivity disorder. Nature Genetics, 51(1), 63.
  6. Hawi, Z., Yates, H., Pinar, A., Arnatkeviciute, A., Johnson, B., Tong, J., ... & Heussler, H. S. (2018). A case–control genome-wide association study of ADHD discovers a novel association with the tenascin R (TNR) gene. Translational Psychiatry, 8(1), 284.
  7. Kuntsi, J., Rijsdijk, F., Ronald, A., Asherson, P., & Plomin, R. (2005). Genetic influences on the stability of attention-deficit/hyperactivity disorder symptoms from early to middle childhood. Biological Psychiatry, 57(6), 647-654.
  8. Kofler, M. J., Rapport, M. D., & Matt Alderson, R. (2008). Quantifying ADHD classroom inattentiveness, its moderators, and variability: a meta‐analytic review. Journal of Child Psychology and Psychiatry, 49(1), 59-69.
  9. Lesch, K. P., Timmesfeld, N., Renner, T. J., Halperin, R., Röser, C., Nguyen, T. T., ... & Freitag, C. (2008). Molecular genetics of adult ADHD: converging evidence from genome-wide association and extended pedigree linkage studies. Journal of Neural Transmission, 115(11), 1573-1585.
  10. Middeldorp, C. M., Hammerschlag, A. R., Ouwens, K. G., Groen-Blokhuis, M. M., Pourcain, B. S., Greven, C. U., ... & Vilor-Tejedor, N. (2016). A genome-wide association meta-analysis of attention-deficit/hyperactivity disorder symptoms in population-based pediatric cohorts. Journal of the American Academy of Child & Adolescent Psychiatry, 55(10), 896-905.
  11. Ollila, H. M., Soronen, P., Silander, K., Palo, O. M., Kieseppä, T., Kaunisto, M. A., ... & Paunio, T. (2009). Findings from bipolar disorder genome-wide association studies replicate in a Finnish bipolar family-cohort. Molecular Psychiatry, 14(4), 351.
  12. Thapar, A., Cooper, M., Jefferies, R., & Stergiakouli, E. (2012). What causes attention deficit hyperactivity disorder?. Archives of Disease in Childhood, 97(3), 260-265.
Figures


Figure 1-Gel Electrophoresis Results of SEMA6D PCR


Figure 2-Chromatogram of heterozygous rs281324 SNP


Figure 3-Gel Electrophoresis Results of SORCS3 PCR


Figure 4-Chromatogram of SORCS3 results


Attention-Deficit/Hyperactivity Disorder (ADHD) affects approximately 8.4% of children (American Psychological Association, 2000). The exact origin of ADHD remains unknown; however, there are theories that ADHD is a result of environmental, neurological, and/or genetic factors (Thapar, Cooper, Jefferies, and Stergiakouli, 2012). This project was developed as a proof of principle to assess the presence of a single nucleotide polymorphisms (SNPs) that was been previously reported to be associated with ADHD. Specifically, the T to C SNP in the SEMA6D (rs281324) and the A to T SNP in the SORCS3 (rs11591402) were examined. The SNP-containing region in the SEMA6D gene was successfully sequenced on 9 de-identified participants, demonstrating feasibility. The region of the SNP in the SORCS3 gene had long stretches of repeat nucleotides so alternative methodologies are needed for this SNP.

Full Paper

Acknowledgments

I'd like to thank Dr. Berish Rubin for his time, guidance, and commitment to mentor the students. A special thank you to Anthony Evans and Devin Rocks for their help in designing primers, and for their time in discussing this case.


This document was last modified 05/20/2019.
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