Amplification of single nucleotide polymorphisms associated with major depression in the aralkylamine N-acetyltransferase (AANAT) gene




Anthony V. Morante

Introduction

Major depression (MD) is one of the major mood disorders (MMD). Typically, those affected by MD exhibit persistent low mood and reduced levels of enjoyment. There have been studies involving twins and families (with biologically related and adopted children) that suggest both genetic and environmental factors play a role in the development of MD (Levinson, 2006). A factor that plays a prominent role in development of MD is the dysfunction of circadian rhythm (Srinivasan et al. 2006). Those affected exhibit disturbances in their sleep/wake cycle, diurnal mood variations, and cyclical recurrence of depressive symptoms.
Melatonin is a regulatory hormone of circadian rhythm that is secreted by the pineal gland. Secretion of melatonin in diurnal mammals remains at a low, constant level during the day and increases in the afternoon at the beginning of the dark-cycle, peaking in the middle of the night. Melatonin targets the suprachiasmatic nucleus in the hypothalamus, which will generate the circadian rhythm (Arendt, 2003).
Aralkylamine N-acetyltransferase (AANAT) is a regulatory enzyme that is involved in circadian rhythm through melatonin synthesis. AANAT is responsible for the conversion of serotonin to N-acetylserotonin through a transfer of the acetyl group from acetyl-coenzyme A to the amine end of serotonin. N-acetylserotonin is then further modified by a methyltransferase to produce melatonin (Hickman et al. 1999). In humans, the AANAT gene it is located in chromosome 17q25. Studies in AANAT-deficient mice revealed increased depressive behaviors, suggesting its role in development of MD (Uz et al. 1999).
In humans, two common single nucleotide polymorphisms (SNPs) are associated with MD (rs3760138 and rs4238989) (Soria et al. 2010). Both SNPs are located in the intron sequence between exons 4 and 5 of AANAT. rs3760138 is a G>T / G>A variant while rs4238989 is a C>G variant. Literature suggests that inheritance of both SNPs leads to increased susceptibility of MD development (Soria et al. 2010).

Materials and Methods

PCR
PCR was performed to target AANAT regions using primers developed flanking regions that contain SNPs (rs3760138 and rs4238989) (Fig. 1a and 1b) as well as in the intronic regions separately flanking exons 2-6 (Fig. 1c). PCR was performed using DNA that was isolated from three de-identified humans and GoTaq Green Master Mix (Promega).
PCR products were visualized using a 1% agarose gel with Ethidium bromide.

PCR Purification
After amplification, products were purified using QIAquick PCR Purification Kit (Qiagen) using manufacturer's instructions.

Sequencing and Analysis
Purified PCR products were sent to GENEWIZ for sequencing.
Sequencing results were analyzed using ApE and FinchTV software.

Results

Amplification of C>G SNP-containing region of AANAT gene
PCR was performed on DNA samples from three de-identified individuals using primers to detect for the C>G SNP (rs4238989) (Fig. 2a, 2b). PCR products of the expected size were generated. The sequencing of the PCR products is displayed in a chromatogram using FinchTV software (Fig. 2c-2e).
The results revealed presence of a guanine and cytosine in the forward sequence at the location of the SNP in each of the samples (Fig. 2c-2e). This pattern was confirmed in the reverse sequence, indicating that this is not an artifact and instead suggests the samples are heterozygous for the SNP (Fig 2c-2e).

Amplification of G>T / G>A SNP-containing region of AANAT gene
PCR was also performed DNA samples from three de-identified individuals using the primers for the G>T / G>A SNP (rs3760138) (Fig. 3a, 3b). PCR products of the expected size were generated. The sequencing analysis using the forward and reverse primers reveal the DNA samples from individuals 1 and 2 are heterozygous for the G>T SNP (Fig. 3c, 3d). The DNA sample of individual 3 revealed homozygous presence of the G nucleotide (Fig. 3e).

Identification of C>T transition in exon region of AANAT gene
The amplified exon sequences were aligned with the database sequences from the NCBI website using ApE software to verify the correct sequence was amplified (Figure 4a-4e). Analysis of exons 2-6 regions of AANAT from individuals revealed only a single SNP in one of the individuals. The SNP is located in the exon 5 region of individual 3, revealing heterozygosity for a C>T transition (Fig. 4f).

Discussion

This proof-of-principle study explores the feasibility of screening for the rs3760138 and rs4238989 SNPs using PCR amplification. The expected PCR products were successfully amplified by the designed primers. This method of screening for SNPs could potentially be used to determining whether an individual possesses the SNPs reported to increase the susceptibility of developing MD.
It is possible that these SNPs are affecting the production of AANAT, which could explain the association of these SNPs to increase susceptibility to MD. However, more studies are needed to explore this possibility.
Additionally, the G>A SNP in rs3760138 results in a GTAAGC sequence, which could potentially introduce an alternative splice donor site as rs3760138 occurs in the intronic sequence of AANAT.


References

1. Levinson DF. 2006. The genetics of depression: a review. Biol Psychiatry;60(2):84-924.
2. Srinivasan et al. 2006. Melatonin in mood disorders. World J Biol Psychiatry;7(3):138-51.
3. Arendt. Importance and relevance of melatonin to humanbiological rhythms. 2003. J Neuroendocrinol
4. Hickman BA, Namboodiri MAA, Klein DC, Dyda F. The Structural Basis of Ordered Substrate Binding by Serotonin N-Acetyltransferase. Cell. 1999 April 30; 97(3):361-369.
5. Uz et al. 1999. Prolonged swim-test immobility of serotonin N-acetyltransferase (AANAT)-mutant mice. J Pineal Res; 30:166-170
6. Soria V, Martinez-Amoros E, Escaramis G, Valero J, Crespo JM, Gutierrez-Zotes A, Bayes M, Martorell L, Vilella E, Estivill X, Menchon JM, Gratacos M, Urretavizcaya M. Resequencing and association analysis of arylalkylamine N-acetyltransferase (AANAT) gene and its contribution to major depression susceptibility. J Pineal Res. 2010;49:3544.



Figures


Figure 1-Diagrams for primer design. (a) Diagram of G>T / G>A SNP amplification. (b) Diagram of C>G SNP amplification. (c) General diagram of exon region amplification.


Figure 2-Identification of G>C transversion in region containing SNP rs4328989. (a) Diagram of AANAT gene structure with C>G SNP (b) Gel image of PCR products for each DNA sample and non-template control (NTC). (c-e) DNA sequences of PCR products. Chromatograms for forward product (top) and reverse product (bottom).


Figure 3-Identification of G>T transversion in region containing SNP rs3760138. (a) Diagram of AANAT gene structure with G>T / G>A SNP (b) Gel image of PCR products for each human DNA sample and non-template control (NTC). (c-e) DNA sequences of PCR products. Chromatogram for forward product (top) and reverse product (bottom).


Figure 4-Amplification of exonic regions 2-6 of AANAT gene. (a-e) Gel image of PCR products for each human DNA sample and non-template control (NTC) (Left) Sequence alignment for amplified product sequence flanking each exon region with actual exon sequence from NCBI website (Right). (f) Identification of C>T transition in exon 5 region of AANAT. Chromatogram for forward product (top) and reverse product (bottom)


Abstract
Disruptions in circadian rhythm are often associated with development of major depression (MD). Aralkylamine N-acetyltransferase (AANAT) is a regulatory enzyme responsible for the synthesis of melatonin, the regulatory hormone of circadian rhythm. Two single nucleotide polymorphisms (SNPs) (rs3760138 and rs4238989) in the AANAT gene are commonly associated with MD development. rs3760138 is a G>T / G>A variant while rs4238989 is a C>G variant. Primers were developed to amplify the regions of AANAT that contain these genetic variations. Using de-identified human DNA samples, the SNP-containing regions were amplified and sequenced to determine whether the samples contained the SNPs. The potential SNP regions were successfully amplified and sequenced in this proof-of-principle study, supporting the use of this method for screening for SNPs associated with MD.

Full Paper

Acknowledgments

I would like to thank Anthony Evans for all of his guidance and generous donation of DNA samples. I would also like to thank Devin Rocks for all of his support during my project. Finally, I would like to thank Dr. Berish Rubin for his help and support to make this project possible.


This document was last modified 05/19/2019.
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