exam questions Flashcards
Question 6 (van Bokhoven; epigenetics, 10 points)
Deletions of chromosome 15q11-q13 can give rise to two distinct conditions. Regardless of the size of the deletion, either Angelman syndrome or Prader-Willi syndrome will be seen in the patient. Explain the underlying mechanisms of the two distinct phenotypes. (10 pts)
Answer:
Both conditions are imprinting disorders. The actual phenotype depends on the parental origin of the deletion. Angelman syndrome is due to a maternal deletion and PWS due to a paternal deletion. In Angelman syndrome one allele is inactivated due to paternal imprinting. The imprinting is the result of DNA methylation AND of the production of a large antisense noncoding RNA. When the other allele has a deletion, no gene product (UBE3A gene) can be made resulting in AS. Conversely, in PWS genes on the maternal allele are repressed due to methylation. When the paternal allele is deleted no gene products are being synthesized, resulting in PWS.
Chat GTP answer:
The underlying mechanisms of Angelman syndrome and Prader-Willi syndrome are different even though they result from deletions of the same chromosomal region 15q11-q13, which contains imprinted genes. Imprinting is a mechanism that leads to the preferential expression of one parental allele over the other in certain genes.
In Angelman syndrome, the deletion or mutation of the maternal copy of the UBE3A gene, which is located in the 15q11-q13 region, is responsible for the syndrome. The paternal copy of UBE3A is normally silenced by imprinting in the brain. However, in some neurons, the maternal copy of UBE3A is not silenced and is responsible for the normal functioning of these neurons. Therefore, the loss of the maternal copy of UBE3A in Angelman syndrome leads to a decrease in UBE3A levels in these neurons, which in turn leads to abnormal synaptic function and cognitive deficits.
In Prader-Willi syndrome, the deletion or mutation of the paternal copy of the same region is responsible for the syndrome. The maternal copy of the imprinted genes is normally silenced in the brain, and it is the paternal copy that is expressed. Therefore, the loss of the paternal copy of the imprinted genes leads to a decrease in the expression of these genes in the brain, which in turn leads to abnormal hypothalamic function and the characteristic symptoms of the syndrome, including hyperphagia and obesity.
Question 3 (Franke; 5 points)
Describe in your own words, what the following statement means for the genetic architecture of the psychiatric disorder ADHD: ‘The disorder ADHD is genetically continuous with traits of inattention and hyperactivity/impulsivity observed in the general population.
ADHD is at the extreme of the distribution of the inattention and hyperactivity/impulsivity traits in the general population, and the same genetic factors contribute to the population traits and the disorder. (5 points; each half of the sentence earns 2.5 points)
ChatGTP answer:
The statement means that the genetic factors contributing to attention deficit hyperactivity disorder (ADHD) are not entirely unique to the disorder but are shared with the general population. In other words, the genetic architecture of ADHD is not dichotomous or black-and-white, but rather it exists on a spectrum that extends into the general population. This suggests that ADHD is not a distinct category but rather a manifestation of normal variation in the population, with more severe cases having a greater number or magnitude of genetic risk factors.
Question 7 (Bralten; 5 points)
Please explain why it can be interesting to study the association of genetic factors of brain phenotypes when interested in the genetics of psychiatric disorders, mention at least two reasons. (2.5 pts per correct answer, max 5 pts)
ANSWER:
-brain phenotypes are phenotypically associated to psychiatric disorders
-genetic correlations are found between brain volume and psychiatric disorders
-effect sizes of genetic risk factors for psychiatric disorders are small, therefore large sample sizes are needed, which can be achieved through large collaborations that in theory can be larger if population-based (brain) phenotypes are used compared to disorder-selected samples.
-brain phenotypes are heritable and hypothesized to lie on the pathway from gene function to psychiatric disorder
ChatGTP answer:
There are several reasons why studying the association of genetic factors of brain phenotypes can be interesting when interested in the genetics of psychiatric disorders:
It can help identify potential causal pathways: Understanding how genetic factors impact brain phenotypes can provide insight into the underlying biological mechanisms of psychiatric disorders. By identifying genetic variants that influence brain structure or function, researchers can gain a better understanding of how changes in brain biology may contribute to the development of psychiatric disorders.
It can improve diagnostic accuracy: Many psychiatric disorders are currently diagnosed based on symptoms alone, which can lead to heterogeneity and imprecise diagnoses. Studying the genetic basis of brain phenotypes associated with psychiatric disorders may provide more objective and reliable measures for diagnosis and may ultimately lead to more targeted and effective treatments.
Question 10 (van Bokhoven, animal models, 5 points)
For a stress-related disorder study in mice you want to investigate whether certain deficits in behaviour originate from altered signalling in between amygdala and insula cortex. Preliminary in vivo data imply that stress-induction increases activity in the amygdala may be core to the behavioural phenotype, but it is unclear whether that is due to hyperexcitability in excitatory or inhibitory neurons. Describe the experimental setting for creating an animal model that would allow cell type specific silencing of network activity in the amygdala using DREADD: Designer Receptors Exclusively Activated by Designer Drugs (5 pts)
Answer
A viral vector construct is needed that expresses an inhibitory DREADD receptor (hM4D1) in neurons. The construct simultaneously expressed a fluorescent protein (GFP, Tomato etc) to identify neurons that have taken up the viral vector. In addition, you can introduce selectively for expression in specific neurons by selection of the right promotor to express the DREADD receptor (e.g. CAMKII for excitatory or GAD67 for inhibitory). These viruses are stereotactically administered to the amygdala of a mouse. Then the mice will either receive a saline (control) injection or injection with the artificial ligand CNO, which will activate hM4D1 and thus silences the neurons in which it is expressed. The effect on stress behaviour can then be analysed (e.g. fear conditioning)
For clarification (not be used as answer) see image below from lecture, Also points will be given when you describe the concept without specific details/names of promotors, or DREADD receptor.
ChatGTP answer:
To create an animal model that allows cell type-specific silencing of network activity in the amygdala using DREADD, the following experimental setting can be employed:
Selection of appropriate DREADD system: 1.Select the appropriate DREADD system for cell type-specific silencing of neurons in the amygdala. There are two types of DREADD systems that can be used - Gi-DREADDs for inhibitory neuron silencing and Gq-DREADDs for excitatory neuron silencing. Select the appropriate DREADD system based on the specific type of neurons being targeted.
2.Design and generation of viral vectors: Design and generate viral vectors that can express the selected DREADD system in the amygdala neurons. The viral vectors can be generated by cloning the DREADD system into a viral vector backbone and packaging it into a virus.
3.Stereotaxic injection of viral vectors: Stereotaxically inject the viral vectors into the amygdala of the mice. This will ensure that the DREADD system is specifically expressed in the neurons of the amygdala.
4.Wait for viral vector expression: Wait for the viral vectors to express the DREADD system in the targeted neurons of the amygdala. This can take around 2-4 weeks.
5.Administration of designer drugs: Administer the designer drug that selectively activates the DREADD system. The designer drug will activate the DREADD system, leading to cell type-specific silencing of neurons in the amygdala.
- Behavioral assays: Conduct behavioral assays to investigate whether the cell type-specific silencing of neurons in the amygdala leads to changes in the stress-related behaviors of the mice. The assays can include tests for anxiety, depression, and other stress-related behaviors.
By employing this experimental setting, it will be possible to investigate whether the stress-induced increase in amygdala activity is due to hyperexcitability in excitatory or inhibitory neurons, and whether silencing of specific types of neurons in the amygdala leads to changes in stress-related behaviors
Question 11 (van Bokhoven, animal models, 5 points)
Mavoglurant, an inhibitor of the metabotropic Glutamate receptor has been used for trials in humans with Fra X syndrome.
a. What was the conceptional rationale for testing this drug? (2.5 pts)
b. Which general problems do account for the lack of translation of promising preclinical studies to patients for neurodevelopmental disorders such as Fra X? Mention four reasons. (2.5 pts)
Answers
a. FMRP is a scavenger of mRNAs. When it is not present there is illegitimate expression of mRNA even in the absence of mGLuR5 activation, which leads to AMPAR internalization and LTD.
b. - Animal models may have more plasticity than humans
- Different pharmacokinetics
- Suboptimal patient selection (age, treatment onset, dosage duration, PK/PD etc
- Most importantly: how to translate a mouse behaviour to a relevant human behaviour
- Placebo effects (strong effects in placebo group, already during run in)
- Other, better predictors of effect needed (biomarkers, biological indicators, cognitive)
ChatGTP answer:
a. The rationale for testing Mavoglurant in humans with Fra X syndrome was based on preclinical studies that suggested that metabotropic glutamate receptor 5 (mGluR5) antagonists could rescue the cellular and behavioural phenotypes in Fmr1 knockout mice, a mouse model for Fra X syndrome. Mavoglurant is a selective antagonist of mGluR5, which is thought to play a role in the pathogenesis of Fra X syndrome.
b. Four general problems that account for the lack of translation of promising preclinical studies to patients for neurodevelopmental disorders such as Fra X are:
Species differences: Preclinical studies are often conducted in animal models, and it is not always clear how well these models recapitulate the human disease phenotype.
Heterogeneity of patient populations: Patients with a particular disorder can have a wide range of clinical features and genetic mutations, which can make it difficult to develop targeted therapies.
Pharmacokinetics and pharmacodynamics: The efficacy and safety of drugs can be affected by factors such as absorption, distribution, metabolism, and elimination, which can be difficult to predict in humans.
Lack of appropriate outcome measures: It can be challenging to design clinical trials with appropriate outcome measures that are sensitive to the effects of the drug and relevant to the patient population. In addition, there may be a lack of consensus on which outcome measures are the most important to use.
Question 1 - Monogenic disorders (van Bokhoven) (10 points)
Fragile X syndrome is caused by a full CGG-trinucleotide repeat expansion at the FMR1 gene, whereas the pre-mutation gives rise to Fragile X Tremor-Ataxia syndrome. View the pedigree below:
A) Why are there no affected individuals in the second generation of this family; i.e. individuals females II.1 and II.3, and the 5 males II.2, II.4-II.7? (3 pts)
B) Explain the more pronounced phenotype in the third generation as compared to the first generation. (3 pts)
C) What is the direct mechanism by which the full mutation the pre-mutation exert their pathological effect. Just explain the molecular consequences, not the effects on cell physiology. (4 pts)
A: Because fragile X is a X linked disorder, so females are rarely affected by it, so that is why the ID in third generation
As the male (I.1) is affected with fragile x tremor-ataxia (prevalent in males), and has this syndrome is an x-linked dominant condition all his daughters but none of his sons are affected. In this case, the daughters are asymptomatic carriers. Probably, in the second generation, the daughters mated with asymptomatic male carriers. This lead to fragile x syndrome in males (as they only have one x) and a milder and variable effect in females (ID) (p66 book genetics)
B: Fragile x disorder appears in generations if is transmitted by the mother so is easier that the grandchildren get the 200 methylated basepairs for getting Fragile x syndrome when the mother carrys the mutated gene, and will get worse with more generations. Only the sons will be affected bc they have only one X chromosome
C: The full mutation makes that there are an excessive amount of CPG islands just before the gene, as a consequence, the islands will be fully methylated and the gene will not be transcribed, so no protein frmes. The premutation has an excess of islands but not enough for not to be transcribed but to affect in general
The fragile X mental retardation 1 (FMR1) gene gives rise to a family of disorders when its non-coding CGG-repeat element is expanded to either the premutation range (55–200 CGG repeats) or the full mutation range (>200 CGG repeats). Although most individuals with the premutation have normal intellectual abilities, some children with the premutation have developmental problems.
The FMR1 (Fragile X messenger ribonucleoprotein 1) gene is located on the long arm of the X chromosome. At the start of this gene lies a region of DNA which varies in length from one person to another. Ordinarily, this stretch of DNA falls within a range of length that would be considered “normal”. In some people, however, this stretch of DNA is somewhat longer; this gene change is a premutation. If the DNA is expanded beyond a certain length, the gene is switched off and does not produce the protein that it normally makes. This gene change is called a full mutation. A male who inherits a full mutation exhibits Fragile X syndrome because his only X chromosome contains the mutated gene.
chat GTP:
A) There are no affected individuals in the second generation of this family because the father (I.1) with the full mutation did not transmit the mutated allele to any of his children in the second generation.
B) The more pronounced phenotype in the third generation as compared to the first generation is due to a phenomenon called anticipation, which is common in Fragile X syndrome. Anticipation refers to the tendency for the severity of the disorder to increase and the age of onset to decrease in successive generations. This is because the repeat length tends to increase during transmission, resulting in more severe disease in subsequent generations.
C) Fragile X syndrome is caused by the expansion of a CGG repeat in the FMR1 gene. In the normal population, the FMR1 gene contains 5 to 44 CGG repeats. Individuals with the full mutation have more than 200 CGG repeats, which results in methylation of the promoter region of the FMR1 gene and subsequent transcriptional silencing. This leads to a deficiency or absence of the FMR1 protein, which is important for synaptic plasticity and cognitive function. In contrast, individuals with the pre-mutation have 55 to 200 CGG repeats, which does not cause methylation and transcriptional silencing of the FMR1 gene. However, the increased number of CGG repeats can interfere with RNA processing, resulting in reduced levels of the FMR1 protein and the development of Fragile X Tremor-Ataxia syndrome.
Question 2 – ADHD genetics (Franke/Klemann) (12 points)
In ADHD, we assume that genetic risk variants affect the brain’s structure and/or function to cause the altered behaviour. Please name and shortly describe three study approaches that can be used to obtain information on the effects of ADHD-relevant genetic variants on the structure or function of the brain. (4 pts for each correct approach and description)
Tip: this can include both in vivo and in vitro study approaches.
Answer:
GWAS: Genome wide association studies, that searches for common variants through the whole genome searching SNPs comparing patients to controls. For this we need a large sample but is the best for finding common variants that are risk factors of ADHD
Brain imaging: Searching differences between cases with ADHD and controls, for example, in brain volume or cortical thickness, and then making correlations with genes that affect ADHD found through other methods like GWAS. With this we can obtain information about the specific pathways of the correlated genes.
Twin studies: We find the heritability, the extent in which genetic individual differences contribute to phenotypic individual differences, using this as a starting point for searching the ADHD mechanisms.
Drosophila: we model ADHD genes in this animal to compare to human phenotype, with this we can confirm the involvement of identified biological pathways
ChatGTP answer:
Genome-wide association studies (GWAS): GWAS are used to identify common genetic variants that are associated with ADHD. By analyzing large datasets of genetic information from people with and without ADHD, researchers can identify specific genetic variants that are more common in those with ADHD. Once these variants are identified, researchers can investigate their effects on brain structure and function using neuroimaging techniques.
Neuroimaging studies: Neuroimaging techniques such as magnetic resonance imaging (MRI), functional MRI (fMRI), and positron emission tomography (PET) can be used to investigate the effects of genetic variants on brain structure and function. By comparing brain scans of people with and without specific genetic variants, researchers can identify brain regions that are affected by the variants.
Animal models: Animal models, such as mice and rats, can be used to investigate the effects of genetic variants on brain structure and function. By creating animal models with genetic variants associated with ADHD, researchers can study the effects of these variants on brain development and behavior. These studies can provide insights into the mechanisms underlying ADHD and may identify potential targets for new treatments.
In vitro studies: In vitro studies involve using cell cultures or animal models to investigate the effects of ADHD-associated genetic variants on brain function. For example, researchers might manipulate the expression of certain genes in animal models of ADHD and then examine the effects on brain structure or behavior. Alternatively, they might use brain tissue from individuals with ADHD to investigate how specific genetic variants affect cellular function in the brain
Question 3 Multifactorial disorders (Arias Vasquez) (10 points)
See the figure below. Trait polygenicity is linked with SNP discoverability. Please describe how the changes in polygenicity affect the susceptibility conferred by SNPs, the individual SNP effect sizes, the expected number of SNPS (density) and our ability to discover them.
Answer:
Changes in polygenicity affect:
Susceptibility conferred by SNPs: More polygenicity, lower susceptibility because the effect of each SNP is very small with high polygenic diseases.
Individual SNP effect sizes: More polygenicity, Less effect size, the trait is affected by more genes that have smaller effect each one, so the effect size of the SNPs will be reduced
The expected number of SNPs (density): More polygenetic, more expected number of SNPs, since more different genes affecting the trait, higher probability of common variants then more SNPs. Expected does not mean that they are easy to find.
Our ability to discover them: More polygenicity, harder to find, since their effect size is smaller, is harder to find significant SNPs and we would need more sample.
LEFT - Rare phenotypes are solely explained by genes (100%). The frequency of these genes is low (because of selection and all)
RIGHT- On the other side there are environmental phenotypes: phenotypes that are explained by the environment (e.g. getting hit by a bus)
MIDDLE: In the middle there’s multifactorial phenotypes: there are environmental and genetic effects. Multifactorial means a combination of smaller effects. Each on itself don’t break system; combination does. There’s also a non-genetic component: environment
Broad-sense model: relation between outcome (disease) and the genes is very complex: it’s like a network
* No direct relation between the phenotype and our genes
* There’s a big difference in how we see genetic effects on traits and diseases:
* (Most) rare diseases: genetic factors are cause for the disease
* In multifactorial diseases: genetic and environmental factors are risk factors for disease
Threshold Model (Falconer)
* Inheritance model of multifactorial disorders
* Liability scale: the amount of load you carry before you become an affected case.
* Accumulation of genetic and environmental effects can cause you to pass a threshold and become affected
* The combination of risk factors (genetic and environmental) above a threshold is necessary for disease manifestation (cumulative effect)
* Accumulation has a requirement (biological condition): individual (genetic) risk factors are of small effect
* Tradeoff between what we want to find vs. how to find: when the effect size is small → we need large sample sizes
Genetic architecture can be summarized by several parameters (Holland et al., 2020; Zhang, Qi, Park, & Chatterjee, 2018): (a) heritability (h2): the overall amount of trait variance explained by genetics; (b) polygenicity (πc): the proportion of susceptibility SNPs (sSNPs), LD-independent loci associated with a trait that are not necessarily genome-wide significant, relative to the total number of LD-independent SNPs in the genome (M); and (c) discoverability (σ): the distribution of effect sizes of sSNPs on a trait. Higher polygenicity of a trait indicates more sSNPs that are associated with that trait (Figure 1). Higher polygenicity is generally associated with lower effect size of each sSNP, requiring higher sample sizes to discover them (Watanabe et al., 2019). Endophenotypes, compared to neuropsychiatric disorders, are hypothesized to have less polygenicity, with greater effect size of each sSNP, requiring lower sample sizes to discover them.
Question 5 – Imaging Genetics (Arenella/Klein) (10 points)
Imaging genetics offers the chance to investigate the genetic underpinning of different brain phenotypes.
a. What is the advantage of applying imaging genetics to psychiatric disorders? (4 pts)
b. Describe a potential approach to use imaging genetics in psychiatry. (6 pts)
Answer:
a. The advantage is that applying imaging genetics we can not only know that genes are related to phenotypes but also get more knowledge about what are these genes exactly affecting, what pathways are they demaging, since we can measure a lot of different types of differences with all the imaging techniques. How genes are risk factors or cause of psychiatric disorders is not well studied yet and imaging genetics gives us the chance of knowing many pathways in detail.
b. In the future, going to the psychiatrist could start with a genetic sequencing which could find a lot of risk factors already related to different brain-phenotypes, that could affect the behavior and the general phenotype of the individual. If we already know the relation, we can have an hypothesis way more accurate than now about what brain parts have problems and improve the diagnostic and treatment of many complex diseases.
ChatGTP answer:
a. The advantage of applying imaging genetics to psychiatric disorders is that it allows researchers to investigate the biological basis of psychiatric symptoms by examining the relationship between genetic variation and brain structure or function. Psychiatric disorders are often complex and heterogeneous, making it difficult to identify specific causes or mechanisms. By using imaging genetics, researchers can better understand how genetic variation contributes to individual differences in brain function and structure, and ultimately, to the development of psychiatric disorders.
b. A potential approach to using imaging genetics in psychiatry involves analyzing large datasets of genetic and imaging data in individuals with and without a specific psychiatric disorder. Researchers can use statistical methods to identify genetic variants that are associated with differences in brain structure or function between these groups. They can also investigate how specific genetic variants interact with environmental factors, such as stress or trauma, to influence brain phenotypes and increase the risk of psychiatric disorders. Additionally, researchers can use machine learning algorithms to identify patterns of genetic and brain imaging data that are predictive of specific psychiatric disorders, which could ultimately lead to the development of personalized diagnostic and treatment approaches.
a. The advantage of applying imaging genetics to psychiatric disorders is that it allows researchers to investigate the biological mechanisms underlying these disorders at a more detailed level. Psychiatric disorders such as depression, anxiety, and schizophrenia are complex and likely involve multiple genes and environmental factors. By using imaging genetics, researchers can identify genetic variants that are associated with changes in brain structure or function, which can help to identify specific pathways or mechanisms that contribute to the development of these disorders. This information can then be used to develop more targeted and effective treatments.
b. A potential approach to using imaging genetics in psychiatry involves analyzing large datasets of genetic variants and brain imaging data in individuals with psychiatric disorders and comparing them to controls. This could involve using techniques such as GWAS to identify common genetic variants that are associated with changes in brain structure or function in individuals with a particular disorder. Researchers can then use these genetic variants as biomarkers to predict the risk of developing the disorder in individuals who are at high risk due to family history or other factors. This information can also be used to develop new treatments that target the specific biological pathways or mechanisms involved in the disorder. Additionally, imaging genetics can be used to identify subtypes of psychiatric disorders that may have different genetic and neurobiological underpinnings, which can help to develop more personalized treatments.
Question 6 – epigenetics and preclinical studies (van Bokhoven) (14 points)
Epigenetic modifications regulate the cell- and tissue-specific expression of our genome. These epigenetic modifications are essential for normal development, and hence, their disruption is often associated with (neuro)developmental disorders
a. X-inactivation and imprinting are driven by epigenetic processes in humans and other mammals. What are the What are the major differences between X-inactivation and imprinting. Name at least 2 differences. (4 pts)
b. The same deletion at chromosome 15q11-q13 can give rise to either Angelman syndrome or Prade Willi syndrome. What is the explanation for this? (4 pts)
c. Currently, several genetic therapies are being developed for treatment of Angelman syndrome. One strategy uses antisense oligonucleotides to “awaken” the the imprinted UBE3A gene. Explain in your own words how this strategy works and what the direct target sequence is of the antisense oligonucleotide. (6 pts)
Answer:
a. One major difference is that X inactivation inactivates the whole chromosome but imprinting ony involves and deactivates only part of the chromosomes. The second difference is that imprinting is always controlled, occurring in the same loci in the whole organism, and X inactivation is Random.
b. Because they are imprinting disorders, Angelman has parental imprinting and PWS has maternal imprinting. The phenotype is caused by the deletion of the gene from the other parent (AS maternal deletion and PWS paternal deletion). In AS, when there is a deletion in the other allele, no UBE3A can be made, resulting in the disease, while in PWS, when the paternal allele is deleted no gene products are being syntesyzed resulting in the disease. Diseases are different because the deletion is in different chromosomes even tough is the same gene.
c. The antisense oligonucleotide targets the gene in the imprinted region, that is imprinted and silenced. We know that the antisense RNA of UBE3A-ATS silences the paternal gene (UBE3A). The Antisense oligonucleotides (ASO) target UBE3A ATS, so the expression of UBE3A occurs in the cells. They use two ASO, ASOA and ASOB for this.
ChatGTP answer:
a. X-inactivation and imprinting are both epigenetic processes that regulate gene expression in mammals, but there are several differences between them. Two major differences are:
X-inactivation occurs in females and involves the silencing of one of the two X chromosomes, while imprinting occurs in both males and females and involves the preferential expression of one of the two parental alleles.
X-inactivation occurs early in embryonic development and is a random process that occurs in each cell, while imprinting occurs later in embryonic development and is established in a parent-of-origin-specific manner.
b. The explanation for why the same deletion at chromosome 15q11-q13 can give rise to either Angelman syndrome or Prader-Willi syndrome is that the deletion affects a region that is subject to genomic imprinting. The genes in this region are normally expressed from only one of the two parental alleles, depending on whether the allele is maternally or paternally inherited. In Angelman syndrome, the maternal copy of this region is deleted, resulting in the loss of expression of several genes, including UBE3A. In contrast, in Prader-Willi syndrome, the paternal copy of this region is deleted, resulting in the loss of expression of several other genes in the same region.
c. One strategy for treating Angelman syndrome involves using antisense oligonucleotides to “awaken” the silenced paternal copy of the UBE3A gene. Antisense oligonucleotides are short pieces of synthetic DNA or RNA that are designed to bind to a specific target sequence in the genome. In the case of Angelman syndrome, the antisense oligonucleotide is designed to bind to the UBE3A antisense transcript, which normally inhibits the expression of the paternal UBE3A allele. By binding to this transcript, the antisense oligonucleotide can prevent it from blocking the expression of the paternal UBE3A allele, thereby increasing UBE3A expression and potentially improving symptoms of the disorder.
Question 7 – Behavioral testing in rodent models (van der Werf) (12 points)
A group of patients has been identified that all carry a loss-of-function mutation in gene X, which has not been linked to any disease so far. The patients present with several neurodevelopmental phenotypes, but only have autism spectrum disorders and anxiety disorders in common. To investigate whether gene X is involved in the disorder of the patients, a knockout mouse model has been developed and is available in your lab for behavioural characterization.
Select one of the behavioural paradigms discussed in the lecture and answer the following questions:
a. Which test did you select? (1 point)
b. Why did you select this specific test? (3 points)
c. Select one of the parameters that can be extracted from this test and describe your hypothesis on how this parameter will be affected in the mouse model for gene X. Illustrate your hypothesis by drawing a graph (include appropriate axis labels and legend) showing the expected result (8 points)
a. Live mouse tracker
b. Because measures Phenotypes typical of anxiety and ASD disorders, like distance travelled (anxiety/ hypo activity), number of interactions and duration of interactions, social behavior, very important phenotype for ASD since they have less social capacities for interaction.
c. Number of specific social interactions: A knockout mice of gene X, will have less social interactions in 24 h than a control. This would tell us a lot about the relation with the human phenotype
Expected result:
E
Chat GTP answer :
a. I selected the elevated plus maze test.
b. I selected the elevated plus maze test because it is a widely used test to assess anxiety-related behavior in rodents. Since the patients carrying the loss-of-function mutation in gene X present with anxiety disorders, this test could provide valuable information about whether the mutation affects anxiety-like behavior in the mouse model.
c. The parameter I selected is the percentage of time spent in the open arms of the elevated plus maze. My hypothesis is that the mouse model for gene X will spend less time in the open arms compared to wild-type mice, indicating increased anxiety-like behavior. I expect this because anxiety disorders are present in the patients with the loss-of-function mutation in gene X. The graph below illustrates my hypothesis.
graph
The x-axis represents the group (wild-type vs. gene X knockout), and the y-axis represents the percentage of time spent in the open arms of the elevated plus maze. The blue bar represents the wild-type mice, and the red bar represents the gene X knockout mice. The graph shows that the gene X knockout mice spend significantly less time in the open arms compared to the wild-type mice, indicating increased anxiety-like behavior in the knockout mice.
Question 8 – Drosophila models (Schenck) (10 points)
Truncating stop mutations in a novel candidate gene for Intellectual disability with autism spectrum disorder have been identified. Describe how you could manipulate the Drosophila orthologue of the gene to address whether it plays a disease-relevant role specifically in neurons.
a) provide a strategy and explain very short what you need to do. (4 pts)
b) name an assay in which you would test the manipulated flies, explain what is measured (no need to explain how exactly the assay is performed) and why this readout is relevant for Intellectual disability/autism spectrum disorder. (6 pts)
Answer:
a. We would make two groups, one Knock out of the gene, and one control. For this we will use a white-eyes mutant control and for the Knock out, a white eyes modified through P-element. We will insert a marker (red eyes) for knowing if the KO worked, and we will take the second generation for choosing the same locus that does not affect anything else. (strain with optimized landing site). Then, the red-eyes will be our KO and the white eyes our control. We will compare their phenotype for ID/ASD.
b. Habituation assay: the jumps are measures, and we check the attenuation of the response to the stimulus (light). This is relevant because most of the genes that are related to habituation are associated with ASD and ID. Drosophila does not model ID/ASD but specific deficiencies, so this could be a nice and easy assay.
Chat GTP answer:
a) One strategy to manipulate the Drosophila orthologue of the gene and investigate its role in neurons would be to generate a neuron-specific knockdown or overexpression of the gene using the Gal4-UAS system. This involves generating transgenic flies that express the Gal4 transcription factor under the control of a neuron-specific promoter, such as the elav promoter, and transgenic flies that contain the UAS sequence upstream of the gene of interest. By crossing these two strains of flies, the Gal4 protein will bind to the UAS sequence in the presence of the inducer, resulting in either knockdown or overexpression of the gene specifically in neurons.
b) One assay to test the manipulated flies could be the courtship assay. In this assay, male flies are placed with a virgin female fly, and their courtship behavior is observed and measured. The readout is the percentage of time spent courting the female, which is relevant for Intellectual disability/autism spectrum disorder because impaired social behavior is a common feature of these disorders. Specifically, in autism spectrum disorder, individuals often have difficulty with social interaction and communication, which can manifest as decreased interest in social interaction, reduced eye contact, and atypical patterns of social behavior. In the courtship assay, if the manipulated flies show impaired courtship behavior compared to control flies, this could indicate a role for the gene in regulating social behavior, and potentially implicate the gene in the pathogenesis of Intellectual disability/autism spectrum disorder.
Question 9 – basic principles (Nadif Kasri) (12 points)
Cofilin is an actin remodeling molecule that potentially plays an important role during long term potentiation (LTP). We know that cofilin is being phosphorylated by CamKII and changes conformation, from an open to closed confirmation, upon phosphorylation:
Design an experiment that will allow you:
a. To find out if cofilin is locally, at the synapse, being activated upon LTP stimuli. (4 pts)
b. To find out the time scale at which cofilin is being activated. What time scale do you expect it to be activated and why? (4 pts).
c. To find out if activated cofilin remains locally or spreads to neighbouring spines. (4 pts)
For all your answers: describe your strategy. Go step by step through the methods you want to apply.
a. To find out if cofilin is locally, at the synapse, being activated upon LTP stimuli, we could use fluorescence microscopy to visualize the subcellular localization of cofilin. Specifically, we could use a fluorescently labeled antibody that binds to phosphorylated cofilin (p-cofilin) to detect the presence of activated cofilin at the synapse. We could then stimulate LTP in hippocampal slices using a high-frequency stimulation protocol and monitor the localization of p-cofilin
Design:
We need to measure coflin and if it is active or inactive, so we will design a coflin that has FRET fluorescence molecules at each end. Then we need to make slices with this probe, and look at the hippocampus. We will use glutamate uncaging and 2-photon imaging that will release the glutamate and mimic the neurotransmitter release. The phosphorylated coflin will be measured before and after LTP.
With this, we can see the time-scale at which coflin is being activated. We expect the time scale to be within the minute range because the initial changes of LTP are in that range (early LTP). Also, we will find out if activated coflin spreads to neigbouringh spines or not.
Chat GTP answer:
a. To find out if cofilin is locally, at the synapse, being activated upon LTP stimuli, we could use fluorescence microscopy to visualize the subcellular localization of cofilin. Specifically, we could use a fluorescently labeled antibody that binds to phosphorylated cofilin (p-cofilin) to detect the presence of activated cofilin at the synapse. We could then stimulate LTP in hippocampal slices using a high-frequency stimulation protocol and monitor the localization of p-cofilin using time-lapse imaging. If we observe an increase in fluorescence intensity at the synapse following LTP induction, this would indicate that cofilin is being locally activated.
b. To find out the time scale at which cofilin is being activated, we could use a similar experimental setup as described above, but perform time-lapse imaging at different time points after LTP induction. We could then quantify the fluorescence intensity of p-cofilin at the synapse over time and determine the time scale of cofilin activation. We would expect cofilin to be activated within seconds to minutes after LTP induction, as cofilin plays a critical role in actin remodeling during the early phases of LTP.
c. To find out if activated cofilin remains locally or spreads to neighboring spines, we could perform fluorescence recovery after photobleaching (FRAP) experiments. Specifically, we could label a subset of synapses with a fluorescent protein, such as GFP, and then photobleach a small region of interest (ROI) at the center of the labeled synapse. We could then monitor the recovery of fluorescence within the bleached ROI over time and determine if activated cofilin spreads to neighboring spines. If we observe a decrease in fluorescence recovery within the bleached ROI and an increase in fluorescence in neighboring spines, this would indicate that activated cofilin spreads to neighboring spines.
Meridelian neurodevelopmental disorders: the example of intellectual disability (van Bokhoven)
You trying to identify genetic risk factors for schizophrenia. Together with a large international consortium (the Psychiatric Genetics Consortium) you have conducted a large genome wide association study (GWAS) study. The GWAS identified more than 100 genomic regions containing genetic variants that were genome-wide significant in the comparison of schizophrenia patients versus controls. Independently, you have used DNA samples from 500 of your patients AND their unaffected parents for exome sequencing.
This revealed about 800 de novo variants associated with schizophrenia. Most de novo variants were unique in the sense that the gene affected by it was not affected by any of the other variants. However, one gene, SETD1A, was affected by a de novo variant in 3 unrelated schizophrenia patients. Notably, the SETD1A locus was not uncovered in the GWAS study.
a. You want to establish the causality of SETD1A variants for the schizophrenia phenotype. What in silico analyses should be done? And what type of genetic studies should be done to collect more evidence? (3 points)
Microarray, reverse FISH analysis, sample will only bind to the mRNA in the complementary wells if it is identical
b. Assume that the SETD1A variants are indeed causative for the phenotype with schizophrenia. Why was this locus not identified in the large GWAS study for which you had contributed all your cases? (3 points)
a. To establish the causality of SETD1A variants for the schizophrenia phenotype, in silico analyses should be done to predict the effect of the variants on the function of the protein. These analyses may include predicting the impact of the variant on protein stability, interaction partners, and functional domains. In addition, functional studies should be conducted to determine the effect of the variants on the protein activity and cellular processes. Genetic studies such as familial segregation analysis and replication in independent cohorts can also be done to collect more evidence.
b. The SETD1A locus may not have been identified in the large GWAS study due to the fact that the de novo variants affecting this gene were not common enough in the general population to reach genome-wide significance in the GWAS analysis. GWAS studies typically require large sample sizes to achieve sufficient power to detect variants that are associated with the phenotype, and rare variants may require even larger sample sizes to achieve statistical significance. Additionally, GWAS studies may have limited power to detect variants with moderate to low effect sizes, which may be the case for SETD1A variants in schizophrenia.
