Sara Samadini, Howard Community College 2017, University of Maryland Baltimore County
Mentored by: Kathryn S. Jones, Ph.D.
Bipolar Disorder (BD) is a common psychiatric illness characterized by recurrent episodes of depression and elevated mood (mania), interspersed with periods of normal mood (euthymia) . However, the clinical picture is more complex. The combination of mood and psychotic symptoms such as delusion and hallucination during the mood swings often makes it difficult to classify the disease and distinguish it from schizophrenia . Various studies have investigated genetic, biochemical, cellular, brain anatomical, and environmental aspects that might contribute to developing BD. In this paper, we will focus primarily on the genetic aspect of the disease.
The genetic code of DNA carries information that determine the structure, function, time of expression, and level of expression of proteins that are the main basis of the cell machinery . The segment of DNA that codes for a specific protein is called a gene. Proteins perform most of the reactions within cells, and are critical for the cell’s structure. Certain proteins control the level of proteins present at any given time, by responding to signals from outside of the cell. Mutations, which are alterations in the DNA sequences within a gene, can lead to disease-causing changes in the structure and/or function of the encoded proteins . Recent studies have shown that many human diseases are the result of mutations in more than one protein.
In addition to genetic factors, environmental factors play a role in many disorders. To investigate the relative contribution of genetic vs. environmental factor in a given disease, many studies apply the use of family and twin studies. Such studies of BD have demonstrated that the heritability of bipolar disorder is considerably high, suggesting that genetic factors are primarily responsible for this disorder. Studies have reported the heritability of BD to be about 85%, which makes it one of the most heritable multifactorial medical conditions . Twin studies have suggested a concordance rate of 0.43 in monozygotic (identical) twins, meaning that the twin of an individual with BD has a 43% chance of also having BD. Population-based family studies have estimated a heritability rate of about 58% indicating that the relative risk in the first-degree relatives is greater than the risk in general population . Such studies indicate that the genetic component in BD is very important.
Scientist have applied different methods and approaches to study the genetics of bipolar disorder such as genome-wide association studies, candidate studies, and phenotype related to BD. A major advance in the ability of scientists to determine which genes encode proteins involved in a specific human disease was the mapping of the entire human genome. Human Genome Project (HGP) was an international effort in which scientists determined the DNA sequence of the entire human genome . Once the genome map was completed, and genomes of many individuals were sequenced, scientists used genome-wide association studies (GWAS) to identify genes involved in human disease. GWAS studies compare large populations to determine variations exist among the population. This method searches for small variations called single nucleotide polymorphism (SNPs) that occur more frequently in people with a particular disease than in people without the disease. These maps have helped scientists to gain a better understanding of the variations in genome of different individuals, functional aspects of the variations, and diseases that might be associated with them.
GWAS and other studies have identified a number of genes that may be suggested to be associated with bipolar disorder. Data from the studies reveals evidence for association of SLC6A4/5-HTT (serotonin transporter gene), BDNF (brain-derived neurotrophic factor), DAOA (D-amino acid oxidase activator), DTNBP1 (dysbindin), NRG1 (neuregulin1), and DISC1 (disrupted in schizophrenia 1) . In this paper, we will focus on the two genes that have been observed to be associated with BD in most of the studies: CACNA1C (calcium channel, voltage-dependent, L type, alpha 1C subunit) and ANK3 (Ankyrin 3). Before discussing how mutation in these genes contributes to the development of BD, we need to have basic knowledge of the anatomy of neurons.
A neuron is a nerve cell and the basic building block of the nervous system. It is specialized to carry messages through an electrochemical process. A neuron consists of dendrites, a cell body (soma), and an axon (Figure 1). Dendrites receive messages from axons of other neurons and send them to the cell body. The message is always sent in one direction, from the dendrites of a cell toward its axon. Two neurons communicate with one another at a junction called a synapse. The neuron that sends the message to the synapse is called the presynaptic neuron and the neuron that receives the message from the synapse is called the postsynaptic neuron. When a message is being sent through the brain, an electrical impulse does not go directly from one neuron to another neuron. Instead, the presynaptic neuron changes the electrical signals to a chemical signal. That chemical signal goes across the synapse, and then is converted back into an electrical impulse in the postsynaptic neuron.
Figure 1: Structure of a neuron 
Terminals of presynaptic neurons contain many synaptic vesicles. These vesicles contain neurotransmitters: the chemical messages that are released into a synapse and received by receptors on the dendrites of the postsynaptic neuron. An electrical message is formed as a result of changes in electrical potential of the neuron. There are different types of protein channels on the membrane of neurons that control the transmission of ions into and out of cells. One of these channels is called the voltage-gated calcium channel. When a presynaptic neuron is activated, the electrical message flows down from the cell body, through the axon, to the axon terminal of the neuron. This impulse activates the voltage-gated calcium channels to open and release the calcium into the neuron’s cytoplasm. This flow of positively-charged calcium ions causes the vesicles to fuse with the cell membrane and release their neurotransmitters into the synapse. These neurotransmitters, then, attach to receptors of the postsynaptic neuron, change the electrical potential of the neuron by activating sodium-potassium channels, and produce an electrical message.
Figure 2: A synapse (“Synapse”) 
The CACNA1C gene is the gene most commonly observed to be associated with BD. That is, specific changes in the DNA of the CACNA1C gene are found more frequently in individuals with BD than those without this disorder. The CACNA1C gene encodes a protein that is part of a type of voltage-dependent calcium channel called CaV1.2. Calcium channels such as CaV1.2 play an important role in ability of cells to make and receive electrical signals. Specifically, CaV1.2 channels are important for normal functioning of brain and the heart. Calcium is usually present at very low levels inside cells, and certain types of proteins are only active when calcium is present. Like other voltage-dependent calcium channels, when CaV1.2 is activated by an electrical signal, it causes a transient increase of the membrane permeability for calcium. This allows calcium into the cell, which activates proteins responsible for causing the vesicles containing the neurotransmitters to fuse with the cell membrane and releasing these neurotransmitters into the synapse.
Since BD is believed to be caused by changes in signaling in the brain, it would make sense that individuals with mutations in a protein that is important for the release of neurotransmitters critical for that signaling would be more likely to have this disorder. However, it is not yet clear how the specific genetic differences observed more commonly in individuals with BD leads to disease. The change in genetic information most highly associated with BD was a SNP called observed at rs1006737. This SNP is located on the third intron of CACNA1C gene, and is hence non-coding . Given the location in a non-coding region of the gene, it is not expected to directly interfere with the structure or functional properties of the CaV1.2 calcium channel. However, variations in non-coding regions often have effects on regulation of expression or splicing of the gene [2,4]. It has previously been shown that there are different versions of the CaV1.2 channel, and that these are produced from the CANCNA1C gene by different patterns of splicing (alternative splicing). Although studies of bipolar disorder have consistently reported elevated basal and stimulated intracellular calcium levels in peripheral blood cells and could have been associated with polymorphisms in CACNA1C, to date none of the SNPs found more commonly found in individuals with BD have been shown to cause these changes. While polymorphisms in CACNA1C are significantly associated with BD, such genetic changes are found to only increase probability of the disease . Studies have indicated that a large number of people who do not have the disease also carry the SNPs in CACNA1C. Thus, the mechanisms regarding how this gene is regulated by rs1006737 SNP or other SNPs, and how changes in this gene contribute to the development of BD, are not yet clear.
Ankyrin 3 (ANK3) gene is another gene with specific genetic changes that are commonly observed to be associated with BD. The exact correlation of ANK3 and development of bipolar disorder is not yet known yet. However, recent findings have shown high ANK3 mRNA level and several SNPs in patients with bipolar disorder compared to healthy individuals. ANK codes for ankyrins, a type of a scaffolding protein that helps to link certain proteins in the cell membrane to part of the cytoskeleton located inside the cell. This allows these proteins, with specific ion channels, to be anchored in the plasma membrane. ANK3 encodes a scaffolding protein (Ankyrin-G) that, in mature neurons, localize to the axon initial segment (AIS). The AIS is a part of the axon where the voltage-gated ion channels, which are critical for sending electrical signals in nerve cells are highly concentrated . The AIS is the part of the axon the closest to the cell body (it is the area in the picture below that pointed to by the word axon).
Figure 3: AIS (“Axon”) 
A number of studies have shown that Ankyrin-G is important for the polarity of neuronal cell, and is particularly important for the AIS. These studies showed that normal functioning of Ankyrin-G is important for transmission of electrical signals by the neurons. The presence of this protein allows voltage-gated sodium channels to cluster, which is important for transmitting the electrical impulse from the cell body through the exon to the axon terminal. Cells with incorrectly functioning Ankyrin-G would not send signals efficiently.
More recent studies have showed that Ankyrin-G also plays an important role in the development of neural cells in the cortex. Ankyrin-G is an important component of canonical Wnt signaling pathway, which plays an important role in development of mammalian central nervous system [3, 7]. Ankyrin-G regulates Wnt signaling by altering the subcellular localization and availability of a protein called β-catenin in proliferating cells . β-catenin is a gene expression regulator protein: a protein that controls expression of other genes. Proper level of β-catenin in the correct place (ventricular zone) of the embryonic brain is required for proper neural progenitor proliferation in the developing cortex . When Ankyrin-G is mutated (and does not function), it increases the level of β-catenin, which changes expression of the protein in a way that promotes progenitor proliferation. In presence of Wnt in the Wnt signaling pathway, a series of reactions happen in the cell which at the end, causes β-catenin in the cytoplasm to enter into the nucleus, produce mRNA, and causes certain Wnt target genes to be expressed, while in absence of Wnt, β-catenin is constantly degraded and the target genes are not expressed. Therefore, Ankyrin-G seems to negatively control the β-catenin level and transcription (gene expression), meaning that individuals with mutated forms of Ankyrin-G cannot properly express certain proteins. Thus, Ankyrin-G may lead to changes in brain development, which might make an individual more likely to develop BD.
The genetics of bipolar disorder is a broad and complicated subject that needs to be investigated in the long-term. The complexity of the disease and the overlaps that it has with symptoms and causes of other psychiatric disorders, not only makes it difficult to distinguish clinically, but also makes it much more difficult to distinguish it genetically. Nevertheless, the disorder is very common, affecting an estimated 50-100 million people world-wide , hence, finding the causes of it is important and requires a great effort.
 An Overview of the Human Genome Project. (n.d.). Retrieved October 25, 2016, from https://www.genome.gov/12011238/an-overview-of-the-human-genome-project/
 Bhat, S., Dao, D. T., Terrillion, C. E., Arad, M., Smith, R. J., Soldatov, N. M., & Gould, T. D. (2012). CACNA1C (Cav1.2) in the pathophysiology of psychiatric disease.Progress in Neurobiology,99(1), 1-14. doi:10.1016/j.pneurobio.2012.06.001
 Durak, O., Anda, F. C., Singh, K. K., Leussis, M. P., Petryshen, T. L., Sklar, P., & Tsai, L. (2014). Ankyrin-G regulates neurogenesis and Wnt signaling by altering the subcellular localization of β-catenin.Molecular Psychiatry,20(3), 388-397. doi:10.1038/mp.2014.42
 Harrison, P. J. (2016). Molecular neurobiological clues to the pathogenesis of bipolar disorder.Current Opinion in Neurobiology,36, 1-6. doi:10.1016/j.conb.2015.07.002
 Kerner, B. (2015). Toward a Deeper Understanding of the Genetics of Bipolar Disorder.Frontiers in Psychiatry,6. doi:10.3389/fpsyt.2015.00105
 Szczepankiewicz, A. (2013). Evidence for single nucleotide polymorphisms and their association with bipolar disorder.Neuropsychiatric Disease and Treatment. doi:10.2147/ndt.s28117
 Valvezan, A. J., & Klein, P. S. (2012). GSK-3 and Wnt Signaling in Neurogenesis and Bipolar Disorder.Frontiers in Molecular Neuroscience,5. doi:10.3389/fnmol.2012.00001
 Zhou, D., Lambert, S., Malen, P. L., Carpenter, S., Boland, L. M., & Bennett, V. (1998). Ankyrin G Is Required for Clustering of Voltage-gated Na Channels at Axon Initial Segments and for Normal Action Potential Firing.The Journal of Cell Biology,143(5), 1295-1304. doi:10.1083/jcb.143.5.1295
 Chand, A. (2010) Grubb Lab; Our Research (For the General Public). Retrieved from http://grubblab.org/For-the-general-public.php
 (n.d.). In Wikipedia. Retrieved October 26, 2016.
 (n.d.). In Wikipedia. Retrieved October 2, 2016.