Copy Number Variations

Copy Number Variants
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Duplications or deletions of genes on chromosomes

Autism spectrum disorder (ASD) is a neuro-developmental disorder that is characterised by three main impairments, social skills, communication, and repetitive behaviours [1]. There is great range in the intensity and symptoms of ASD and thus there is no exclusive cause that has been determined [1]. However, deletions and duplications in sections of chromosomes, called copy number variants (CNVs), have demonstrated promising correlations to the causation of ASD [3]. The following CNVs, CAPS2, PTCHD1, and NRXN1, have been proposed to have effects on synaptic adhesion proteins, the release of brain-derived neurotrophic factor, and cerebellar deficits among others [1] [2] [3].

1 CAPS2 on Chromosome 7q31.32

Calcium-dependent activator protein for secretion 2, or CAPS2, is found on or around the vesicles in the presynaptic terminals of granule cells which connect to the postsynaptic spines of purkinje cell dendrites [4]. The gene that encodes CAPS2 is located on chromosome 7q31.32, which is within the locus associated with autism susceptibility, AUTS1[5]. Through animal model experiments CAPS2 has been found to be associated with many behavioural phenotypes of autism spectrum disorders, such as an impairment in social activity, hyperactivity, as well as anxiety and a reduction in exploration of a new environment[6]. These behavioural deficits are believed to be the consequence of reduced secretion of BDNF and an underdeveloped cerebellum[5].

CAPS2 and BDNF Secretion in Neurons
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(a) CAPS2 anchored on secretory vesicles aids in the secretion of BDNF
(b) CAPS2 knockout shows an impairment in BDNF secretion
(Sadakata & Furuichi, 2010)

1.1 CAPS2 Decreases BDNF Secretion

It has been demonstrated that mice with a homozygous knockout of the CAPS2 protein show a decrease in the secretion of brain-derived neurotrophic factor, BDNF [6]. This decrease in BDNF secretion is due to the fact that CAPS2 is located on secretory vesicles, and therefore when there is an absence of CAPS2, the vesicles containing BDNF are unable to be exocytosed [7]. Considering that BDNF is not released, none of its downstream effects are able to occur, as seen in figure 1 [9]. BDNF’s downstream effects are associated with the growth, survival and differentiation of neurons and synapses [8].

In a study performed by Washida et al.(2007), the CAPS2 gene was deleted by removing a part of the first exon. This action resulted in a mouse with homozygous deletion of the CAPS2 protein. The CAPS2 knockout mouse was then tested against a wild-type mouse to ensure that all of their cognitive and sensory abilities remained intact [6]. These tests included visual, auditory, and olfactory analysis, as well as the classic Morris water maze test [6]. Motor function and initial performance of the knockout mice was indistinguishable from that of the wild-type mice [6]. However, when the hidden platform in the water was removed, the knockout mice showed an impairment in spatial accuracy in locating where the platform was supposed to be [6]. Therefore, this shows that the CAPS2 knockout mice have an impairment in spatial memory retention [6].

Results of Behavioural Tests for CAPS2 Knockout Mice
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(A) number of interactions with novel mice. The black bar representing CAPS2 -/- shows a significant decrease.
(B) Activity in a cage after habituation. CAPS2 -/- show an increase.
(C) Open Field Test without a novel object. CAPS2 -/- decrease in activity.
(D) Open Field Test with a novel object. CAPS2-/- decrease in activity. (E) (F) Movement of the mice during the Open Field Test.
(Washida et al., 2007)

Washida et al. (2007) then tested both types of mice with examinations that would reveal behavioural characteristics associated with autism, such as social impairments, hyperactivity, or a decrease in exploration of a new environment or object. One such examination involves placing a mouse into a cage with another mouse that it has never met before [6]. The results demonstrate that homozygous CAPS2 deletion mice spent less time interacting with the novel mouse in comparison to the wild-type mice, who frequently showed signs of interaction with the novel mouse [6]. Other results included, high anxiety levels and, in comparison to wild-type mice, a lower tendency to explore a new environment as seen in the classic open field test [6]. Furthermore, the CAPS2 knockout mice were less likely to observe, or make any form of contact with a new object in the novel object test, and they were shown to have reduced locomotor activity in the 8-arm radial maze test [6]. One of the most important social behaviours exhibited by mice and humans alike is maternal care [6]. Interestingly, the pups of the knockout mice had a lower rate of survival due to the neglect of the mothers [6]. In order to ensure that it was the mother that possessed the lack of nurturing, and that there was no relation to the pups, the experimenters took the pups born to the knockout mice and gave them to the wild-type mice to nurture [6]. The pups nurtured by the wild-type mice had a normal survival rate, which implicates that CAPS2 knockout mice do have a severe lack in social behaviour [6]. With all of these examinations, it is clear that CAPS2 knockout mice possess qualities and behaviours that mimic those of autistic individuals, due to the lack of BDNF secretion and thus lack of proper neuronal and synaptic growth [6].

A novel paper by Sadakata et al. (2013) shows the importance of the CAPS2 gene by way of heterozygous knockout mice [5]. They used these knockout mice in a number of behavioural tests similar to those in the Washida et al (2007) paper, such as the open field test and novel object recognition test, and tested them against wild type mice to discover the behavioural deficits [5]. The researchers were able to show that due to the loss of BDNF release from neurons because of the reduced CAPS2 protein, the CAPS2 heterozygotes were unable to adapt to novel environments, and showed an impairment in ultrasonic vocalization to pups [5]. The disabilities involved with the incomplete CAPS2 protein expression suggest its importance in the onset of autism spectrum disorder [5].

1.2 CAPS2 effect on cerebellar development

In terms of brain structures that correspond to the deletion of the gene for CAPS2, there are no large abnormalities from wild types [10]. There is, however, a small fissure missing between two lobules (VI and VII) in the cerebellum [12] of the CAPS2 mice which is not present in wild-type mice [10]. In addition, there are severe developmental issues for cerebellar neurons, including differentiation and survival of said neurons, as well as drastically reduced length in Purkinje cells [9]. It has been demonstrated that autistic patients have a deficit in motor function due to the underdevelopment of the cerebellum [13]. An experiment to discover the development of the cerebellum was performed on mice with the CAPS2 knockout gene [10]. In control mice, massive proliferation of granule cells occurs in the external granule layer of the cerebellum, and Purkinje cell dendrites grow extensively [10] [11]. In order for this to occur, BDNF must be released by granule cells so as to bind to Trk receptors, TrkB and TrkC, on Purkinje cells and granule cells [10]. Once the binding occurs, Trk signalling commences which is vital for the survival and differentiation of the cells [10]. Thus, since removing CAPS2 causes a lack of BDNF release, the CAPS2 knockout mice showed a reduction in Trk signalling and consequently a decrease in Purkinje cell and granule cell differentiation and an increase in apoptosis [10].

In order to test the function of the cerebellum in CAPS2 knockout mice, rotarod performance tests were completed [10]. In mice that have motor deficits there is an increase of slippage and falls on the spinning rod, in comparison to mice with no cerebellar issues [10]. Therefore, CAPS2 not only plays a role on the cellular level but also on the functional level of the cerebellum.

Cerebellar Fissure
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Fissure in between lobules VI and VII in the CAPS2-/-
in comparison to the CAPS2+/+
(Sadakata, T., Kakegawa, W., Mizoguchi, A., Washida, M., Katoh-Semba, R., Shutoh, F., et al., 2007)

1.2.1 PTCHD1 Gene’s effect on the cerebellum

Human Patched domain-containing protein 1, or PTCHD1, is a deleted gene on chromosome Xp22.11 [1]. PTCHD1 is a transmembrane protein that possesses a patched-related domain, which in turn, is related to Hedgehog receptors [15]. Hedgehog is a signalling pathway that is essential for the formation of the neural tube, and motor neuron differentiation [1] [15]. Expression of the PTCHD1 gene is generally found to be in the cerebellum, and is highly associated with other cerebellar genes such as CAPS2 [1]. In a study performed by Noor et al. (2010), a number of families with autism spectrum disorder were screened for the PTCHD1 gene. Each of the family members with ASD were shown to have a mutation of the PTCHD1 gene, and this was revealed to be associated with the autistic phenotype of learning and developmental disabilities, as well as cerebellar abnormalities [1] [14]. Therefore, PTCHD1 is also a good candidate gene for autism, along with CAPS2, since it causes many autistic phenotypes [1].

2 NRXN1 gene

Of all human genes, the neurexin-1 (NRXN1) gene is one of the largest [16]. The NRXN1 gene resides on chromosome 2p16.3 and is important for synaptic formation and maintenance, such as aligning pre-synaptic neurons to post-synaptic neurons to ensure proper communication of neurotransmitters [16][17].

2.1 Role in synaptic formation and maintenance

Location of Neurexin in the Synapse
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Neurexin aligns pre and post synaptic neurons for proper neurotransmitter communication [16]
(Reichelt, Rodgers, & Clapcote, 2011)

In order for neurons in the brain to be able to communicate with each other, neurotransmitters must be released into synapses [16]. Vesicles in the pre-synaptic axon release neurotransmitters, which bind to specific receptors on the dendrites of the post-synapse [16]. Effective communication in the synapse is achieved through alignment of the vesicles and receptors [16]. Cell adhesion using neurexins and neuroligins is necessary for alignment and synaptic formation [16]. Therefore, neurexins are essential for communication [16].

Neurexin-1α, a subset of the NRXN1 gene, is required for the release of neurotransmitters by way of calcium [16]. In an experimental study by Missler et al. (2003), mice with a neurexin-1α deletion were examined [16][18]. It was found that these mice had impairments in neurotransmitter release due to calcium channel malfunction, although there were normal levels of cell-surface channels [16][18]. Although the knockout mice demonstrated a loss of synapse function, there was no loss in synapse numbers or change in structure, which suggests that neurexin-1α is fundamental for the maintenance and function of synapses, but not necessarily for the initial formation of the synapses [16][18].

In addition to the mouse model, neurexin-1 knockout Drosophila flies were examined [19]. An experiment was performed with the purpose of discovering the viability, fertility, and learning ability of the Drosophila [19]. First, the experimenters created neurexin-1 null mutants and compared them to wild-type Drosophila [19]. The neurexin-1 knockouts were shown to be viable and fertile, identical to the wild-type, however, the knockouts exhibited a lower survival rate [19]. Less than half of the neurexin-1 knockout Drosophila survived past 37 days, in comparison to the wild-type in which almost 85% survived [19]. Next, the learning ability of both types of Drosophila was assessed through an associative learning task using scents paired with a fructose reward [19]. The neurexin-1 null mutants demonstrated a deficit in this learning task, which is likely due to the fact that there is a decrease in synaptic function and formation caused by the neurexin-1 deletion [19]. The lack of associative learning seen in the neurexin-1 knockout Drosophila is also seen in patients with autism, which further demonstrates the importance of the neurexin-1 gene [19].

2.2 Phenotypic effects

Autistic individuals may have a variety of phenotypes that deal with impairments in communication, social behaviour, and restricted or repetitive behaviour [1]. Adding to the phenotypic effects already mentioned, Etherton et al. (2009) conducted a series of classic tests on neurexin-1 knockout mice and wild-type mice in order to determine if they possessed some of the same traits as those with autism [20]. The results revealed decreases in synaptic strength, prepulse inhibition, nest building, and maternal care [16]. The nest building and maternal care deficits are analogous to the social issues observed in autistic patients, which demonstrate that the neurexin-1 knockouts have difficulties in caring for themselves and others [20]. Due to the fact that patients with ASD have shown impaired prepulse inhibition, it was tested whether or not the knockout mice showed the same behaviour [20]. Moreover, patients with the NRXN1 deletion who were also diagnosed with autism were shown to have developmental delays in areas such as walking and talking [3]. Together, all of these studies suggest the great importance of the neurexin-1 gene in autism.

2.3 Paternal VS maternal inheritance

Autism affects approximately 3-6 births out of every 1,000 and there is a ratio of 3 males: 1 female [6] [23]. Furthermore, twin studies have shown that if one dizygotic twin is autistic there is a 90% chance the other twin will also be autistic [6]. However, for monozygotic twins there is only a 10% chance, implying that there is a strong genetic component underlying autism [6]. The NRXN1 gene is thought to be inherited recessively by either the mother or father [21], and multiple studies have supported this conclusion [3] [17] [22]. Considering that the majority of the parents of the autistic children with NRXN1 deletions were unaffected, it is suggested that the deletion is not fully penetrant [16]. With this, it has been proposed that although the neurexin-1 gene seems to be highly involved in the genetics of autism, that it is dependent upon penetrance and that there are additional genetic variants [24].

1. Noor, A., Whibley, A., Marshall, C., Gianakopoulos, P., Piton, A., Carson, A., et al. (2010). Disruption at the PTCHD1 locus on Xp22.11 in autism spectrum disorder and intellectual disability. Science Translational Medicine, 2(49), 1-16. Retrieved March 4, 2013, from the Pubmed database.
2. Sadakata, T., Kakegawa, W., Mizoguchi, A., Washida, M., Katoh-Semba, R., Shutoh, F., et al. (2007). Impaired cerebellar development and function in mice lacking CAPS2, a protein involved in neurotrophin release. The Journal of Neuroscience,27(10), 2472-2482. Retrieved March 4, 2013, from the Pubmed database.
3. Schaaf, C., Boone, P., Sampath, S., Williams, C., Bader, P., Mueller, J., et al. (2012). Phenotypic spectrum and genotype-phenotype correlations of NRXN1 exon deletions. European Journal of Human Genetics, 20, 1240-1247. Retrieved March 4, 2013, from the Pubmed database.
4. Sadakata, T., Mizoguchi, A., Sato, Y., Katoh-Semba, R., Fukuda, M., Mikoshiba, K., et al. (2004). The secretory granule-associated protein CAPS2 regulates neurotrophin release and cell survival. The journal of neuroscience , 24 (1), 43-52.
5. Shinoda, Y., Sadakata, T., Oka, M., Sekine, Y., & Furuichi, T. (2013). Autisitic-like behavioural phenotypes in a mouse model with copy number variation of the CAPS2/CADPS2 gene. Federation of European Biochemical Societies Letters , 587 (1), 54-59.
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