Midterm 2 Flashcards
(390 cards)
1
Q
AAllelic heterogeneity definition and example
A
The presence of multiple alleles at a single locus resulting in the same phenotype/disease
e.g. CTFR protein is an ion channel that helps to maintain the balance of salt and water on many surfaces, like lungs and gastrointestinal tract.
- There are over 1,000 mutant alleles of the CTFR gene that causes cystic fibrosis (mucus layers that can attract infections due to ion imbalance)
2
Q
3 examples of mutations that result in allelic heterogeneity
A
- Hypomorphic allele
- Null allele
- Novel property allele
3
Q
Hypomorphic vs. null allele examples of allelic heterogeneity
A
Hypomorphic allele (partial LOF): results in milder phenotype
Null allele (complete LOF): severe phenotype/complete set of phenotypes
4
Q
Example of novel property allele example of allelic heterogeneity
A
Sickle cell disease mutation in beta-globin (Glu6Val) -> clumped hemoglobin at low oxygen conditions
(as opposed to the loss of function mutation in beta-globin gene HBB -> beta-thalassemia)
5
Q
Locus heterogeneity definition and example
A
The phenomenon in which mutations at different genetic loci cause a similar phenotype
e.g. retinitis pigmentosa
- mutations in >100 genes can independently cause retinities pigmentosa (has autosomal dominant, autosomal recessive and X-linked origins)
- Only one mutant locus is needed for the phenotype to be manifested
6
Q
Modifier gene definition and
CF example
A
A gene that modifies the effect produced by another gene
- e.g. although cystic fibrosis is mostly caused by mutations in CTFR gene, CF patients have highly variable lung disease due to CTFR modifier genes like mannose-binding lectin, glutathione-S-transferase, transforming growth factor-beta1, tumor necrosis factor-alpha, etc (the CTFR protein is heavily modified after translation, so mutations in any of the enzymes that modify the protein can impact cystic fibrosis manifestation)
7
Q
Modifier gene example in cancer
A
Mutations in BRCA1 and BRCA2 genes increase the risk of developing breast and ovarian cancers, while mutations in the CHEK2 gene further accelerate this risk
8
Q
What are two examples enzymatic defects and their associated diseases?
A
- Phenylalanine hydroxylase (disease: PKU)
- Adenosine deaminase (disease: severe combined immunodeficiency)
9
Q
True or false: PKU is irreversible
A
False; if detected in newborn within 4 weeks of birth, this disease can be completely reversed.
10
Q
Enzymopathy definition and example
A
A metabolic disorder resulting from deficiency or abnormality of a specific enzyme
- e.g. Hyperphenylalaninemias refer to a group of disorders characterized by elevated levels of phenylalanine in the blood, ranging from mild to severe. Phenylketouria (PKU) is a specific form of this condition.
11
Q
Hyperphenylalaninemia metabolic result, visible phenotype and causes (2)
A
Elevated blood level of amino acid Phe, which we obtain from diet.
- can cause: intellectual disability, seizures, and behavioural issues
- an autosomal recessive disorder due to the deficiency of enzyme phenylalanine hydroxylase (PAH) or cofactor tetrahydrobiopterin (BH4), which is a coenzyme in the enzymes involved in Phe processing
12
Q
Phenylalanine hydroxylase converts phenylalanine (essential/non-essential) amino acid into _____ (essential/non-essential)
A
Essential, tyrosine (non-essential)
13
Q
What is phenylalnine hydroxylase important for?
A
Involved in the pathway producing dopamine (Tyr is converted into dopamine)
14
Q
What 3 pathways are disrupted when BH4 is deficient?
A
- Conversion of Phe into Tyr by Phe hydroxylase
- Conversion of Tyr into dopamine by Tyr hydroxylase
- Conversion of Trp into serotonin by Trp hydroxylase
15
Q
Classic PKU severity, cause and treatment
A
Severe form of disease (elevated [Phe] damages the developing central nervous system in early childhood and interferes with the function of the mature brain, resulting in mental retardation
Cause: deficient PAH
Treatment: low-phenylalanine diet
16
Q
Variant PKU severity, cause and treatment
A
Partial disruption of enzyme (intermediate severity)
Cause: deficient PAH
Treatment: low-phenylalanine diet (less restrictive than that required to treat classic PKU)
17
Q
Non-PKU hyperphenylalaninemia severity, cause and treatment
A
Disruption is so mild that generally they don’t have to be treated
Cause: None, or less restrictive low-phenylalanine diet
18
Q
Mutation(s) that results in deficient PAH, and what causes different phenotype severity
- Explain what type of genetic heterogeneity this is
A
Gene: 12q24.1
Range in severity due to allelic heterogeneity
19
Q
Impaired BH4 recycling mutation(s) and treatment, and what causes different phenotypes
A
Two loci (locus heterogeneity):
1. 10q22 - low-phenylalanine diet + L-dopa, 5-HT, carbidopa
2. 4p15.31 - low-phenylalanine diet + L-dopa, 5-HT, carbidopa + folinic acid
20
Q
True or false: BH4 deficiencies are more common that PAH deficiencies
A
False; BH4 deficiencies are rare
21
Q
What 2 enzymes are affected in impaired BH4 recycling?
A
PCD and DHPR
22
Q
Impaired BH4 synthesis mutation(s) and treatment, and what causes different phenotypes
- Explain what type of genetic heterogeneity this is
A
Two loci (locus heterogeneity):
1. 14q22
2. 11q22.3-23.3
Same treatment for both: low-phenylalanine diet + L-dopa, 5-HT, carbidopa + folinic acid, and pharmacologic doses of BH4
23
Q
What 2 enzymes are affected in impaired BH4 synthesis?
A
GTP-CH and 6-PTS
24
Q
True or false: as with PKU caused by PAH deficiency, a change in diet is enough to treat those with BH4 deficiency
A
False; diet isn’t enough because BH4 mutations disrupt many functions and not just PAH
25
Classic PKU inheritance pattern?
Autosomal recessive
26
True or false: the exact neuropathological mechanism for how Phe causes toxicity is unknown
True
27
PKU frequency in live births
1/2,900
28
Describe how newborns are tested for PKU
PKU is the prototype of genetic diseases for new born screening
- The screening test is performed a few days after birth by taking droplets of blood from a heel prick to analyze the ratio [Phe]/[Tyr]
- Positive results must be confirmed quickly because delayed initial treatment beyond 4 weeks after birth has profound effects on the intellectual outcome of patients with PKU
29
True or false: variant PKU and non-PKU hyperphenylalaninemia are less severe than the classic PKU
- explain why or why not
Due to the residual activity of the mutated PAH enzyme
30
How many different mutations in the PAH gene (alleles) have been identified worldwide? Most PKU patients are...
>400 different mutations
- Most PKU patients are compound heterozygotes (i.e. with 2 different mutant alleles at the same locus)
31
What % of hereditary hyperphenylalaninemia patients have wildtype PAH? What causes their PKU phenotype?
1-3%, defected genes involved in the formation or recycling of the PAH cofactor, BH4
32
A mother with homozygous LOF mutation in PAH gene gives birth to a child that is heterozygous for that mutation in PAH. However the child still develops PKU symptoms because...
The mother was not treated with a low-phenylalanine diet and the child is exposed to high levels of phenylalanine from the mother's blood circulation. The high levels of Phe that disrupt the nervous system is what causes the PKU phenotype, it doesn't matter where they came from.
33
What 3 enzymes require BH4, that become defective in PKU patients with defective BH4?
1. PAH
2. Tyrosine hydroxylase (converts Tyr into L-dopa -> dopamine -> NE and E)
3. Tryptophan hydroxylase (converts Trp to 5-OGH trp -> serotonin)
34
What testing was done before 1960 and since 1990s to test for PKU in newborns?
Gurthrie test since 1960s and tandem mass spectrometry since 1990s
35
True or false: PKU was the first disorder to benefit from newborn screening
True
36
Definition of immunogenetics and what the field explores
Definition: A branch of biology that studies the genetics of the immune system
- Immunogenetics explores the intraspecific variability, tissue-receptor genetics (tightly regulated process), cancer immunology (trying the prime the immune system against a tumour, which would be less harmful than chemo), and the genetic and population-level aspects of host-pathogen interactions, as well as issues surrounding tissue compatibility (with organ donations)
37
How many immune cells are there? How much do they weigh in total?
1.2-1.5 trillion immune cells = ~1.2 kg
38
How many immune cells are freshly generated each day?
1 to 2 billion immune cells
- rapid turnover
39
Definition of immune response
Antigen has been recognized, and body is trying to do something against it
40
Cellular vs. humoral immune response
Cellular = T cells, B cells
Humoral = antibodies (anti-microbial peptides)
41
Describe the relationship with innate immunity/adaptive immunity and birth
- Innate immune cells, and innate immunity are always present in the body from birth
- Adaptive immune cells are present at birth, but immune specificity develops after exposure to specific foreign substances
SO BOTH TYPES OF IMMUNE CELLS ARE PRESENT AT BIRTH BUT ADAPTIVE IMMUNE RECOGNITION SYSTEM ISN'T THERE AND EXPOSURE TO DIFFERENT PATHOGENS OVER LIFE TRAINS THE ADAPTIVE IMMUNE RESPONSE
42
What 7 cell types are considered to be innate immune cells?
Platelets, eosinophils, basophils, neutrophils, monocytes, macrophages, erythrocytes
43
What cells do innate immune cells originate from?
Myeloid progenitor cells
44
What 2 cells are considered to be adaptive immune cells?
T-cells and B-cells
45
What cells do adaptive immune cells originate from?
Lymphoid progenitor cells
46
Describe the specificity of response in innate immunity vs. adaptive immunity
- Innate immunity is non-specific, meaning that it can respond to any foreign invader without discrimination (they just create a toxic environment)
- Adaptive immunity is highly specific, targeting only particular pathogens it has encountered or has been exposed to (including vaccines)
47
Describe the speed of response in innate immunity vs. adaptive immunity
- Innate immunity is rapid (first line of defense), acting immediately to earl hours upon encountering a pathogen.
- Adaptive immunity takes longer to get activated, typically 1-2 weeks, as it needs to recognize the pathogen and mount a tailored response.
48
Describe the composition in innate immunity vs. adaptive immunity
- The innate immune system comprises several cells (i.e. immune epithelia) and chemicals (ROS). Immune cells: phagocytic leukocytes (neutrophils and macrophages), dendritic cells, natural killer cells.
- The adaptive immune system, on the other hand, consists of B cells (produce antibodies) and T cells (destroy infected host cells or help activate other immune cells)
49
Describe the development and evolution in innate immunity vs. adaptive immunity
- Innate immunity is evolutionarily older, found in both vertebrates and invertebrates, providing a basic level of defence
- Adaptive immunity is more recently developed, exclusive to vertebrates, and offers a highly specialized defence through memory and specificity
50
Describe the origin and differentiation of the adaptive immune cells
Lymphoid stem cells originate in the bone marrow, and differentiate into T cells in the thymus and B cells in the bone marrow
51
In general, how do B and T cells develop diverse cell-surface receptors?
Through VDJ recombination and junctional diversity
52
Describe the migration and antigen encounter of the adaptive immune cells
Post-development, these cells circulate to secondary lymphoid organs (e.g. spleen, lymph nodes)
- Encounter foreign antigens, usually processed by antigen-presenting cells (APCs) for T helper cells
53
In the adaptive immune response, what is the antigen first recognized by? What do these cells do and what does this allow for?
Antigen-presenting cells (APCs).
APCs engulf some part of the pathogen, and class II MHC molecules on APCs transport foreign peptides to the cell surface. This allows for the adaptive immune system (helper T cells) to recognize the pathogen.
- APCs play a key role in bridging the innate and adaptive immune responses
54
What 3 cells are considered to be professional APCs?
Dendritic cells, macrophages and B cells.
55
What do helper T cells do?
Upon recognition of the foreign peptide presented by class II MHCs via T-cell receptors, they secrete cytokines that are toxic proteins.
56
What are the functions of cytokines?
Secreted by helper T cells, they provide a toxic environment and they stimulate B cells with immunoglobulins specific to the foreign peptide
57
How are B cells "double validated" upon infection?
They are stimulated by cytokines released from helper T cells, and they recognize the antigen independently through antigen binding with its receptors: B cell receptors (BCRs), membrane bound immunoglobulins
58
B-cell receptors are part of which family of proteins, and become what once they detach?
Are in the immunoglobulin family, they become antibodies once they're detached from the cell.
59
True or false: the immune system creates equal numbers of different types of B cells
True
- Immune system produces a bunch of B cells because it knows that it will be exposed to millions of different entities, and it knows that each one of them could be potentially lethal
60
In general, what process enhances B-cell receptor diversity and affinity for a foreign antigen?
Somatic hypermutation upon B cell binding to antigen
61
What does activation of the adaptive immune system result in, in general?
Activation leads to a humoral (B cell) and/or (T cell) immune response against the foreign antigen
62
Activated B cells are also known as...
- How are these cells activated?
Plasma cells
- Called this once the antigen has been recognized by a B cell and the B cell is mounting an immune response by releasing antibodies specific to the antigen
63
What happens to B cells after an infection?
Stimulated B cells differentiate into plasma cells that secrete antibodies (secreted immunoglobulins) into the circulation, which bind to the antigen (e.g. component of a microbe), thereby neutralizing and eliminating infection
64
What do T-cell responses include upon activation of the adaptive immune response?
T-cell responses include cytotoxic T cells (destroy infected cells), regulatory T cells (modulate the immune response by shutting down certain T cells), and memory T cells (quick response to future encounters with antigen
65
Where do B cells get their name from?
The bursa of Fabricius, a lymphoid organ in birds where B cell maturation was first studied.
66
Where do T cells get their name?
The thymus, where they mature
67
Which of the following best describes the presence of innate and adaptive immunity in the body?
a) Innate immunity develops after birth, while adaptive immunity is present at birth
b) Both innate and adaptive immunities are present from birth
c) Innate immunity is mostly developed by birth, while adaptive immunity mostly develops after exposure to specific foreign substances
d) Adaptive immunity is always present from birth, while innate immunity develops over time
e) Both innate and adaptive immunities develop after exposure to pathogens
C
68
What are the three areas where the diversity of the adaptive immune response comes from?
1. Different B-cell receptors
2. Different T-cell receptors
3. Different MHCs
69
What chains do antibodies consist of?
2 identical light chains and 2 identical heavy chains.
70
What region(s) is/are found on the light chains of an antibody?
The variable, joining and constant regions
- Joining region joins the variable and constant regions
71
What region(s) is/are found on the heavy chains of an antibody?
The variable, joining, constant and diversity regions. The diversity region is an additional smaller peptide on heavy variable chains, located between its variable and joining regions
72
True or false: each region on the antibodies is produced by a different gene
True
73
How many distinct antibodies can B cells generate?
~100 billion
74
What was the one gene-one antibody hypothesis? How was this disproven?
Each gene only coded for one antibody.
- Disproven because only 25,000 protein-coding genes in the human genome
75
How many V genes are there in the human genome?
More than 80V segments for light and heavy chains
76
How many J genes are there in the human genome?
More than 6 J segments for light and heavy chains
77
How many D genes are there in the human genome?
About 30D gene segments for heavy chains
78
True or false: There's only 1 V, 1 J and 1 D gene per antibody
True
79
What process in general produces a vast repertoire of antibodies and T cell receptors?
V(D)J recombination creates 100,000 to 1,000,000 antibody types through DNA sequence deletion and rearrangement
80
What is junctional diversity in antibodies?
Variability at V,D, and J joining points due to nucleotide insertions/deletions
81
True or false: V(D)J recombination is the same thing as splicing, but for antibodies
False; V(D)J recombination is not the same as splicing because splciing happens in pre-mRNA
82
What part of the antibody provides specificity to antigen binding?
The variable region (which binds to the antigen)
83
Where does V(D)J take place?
In the bone marrow for B cells and Thymus for T cells
84
Describe somatic hypermutation
When the antibody first binds to the antigen, somatic hypermutation is kickstarted. This now starts to produce hundreds of versions of the B cell clone through high mutation rates in V segment genes. Some clones start losing affinity (lost in system) and some start gaining affinity
85
Other than junctional diversity, V(D)J recombination and somatic hypermutation, what contributes to the diversity of antibodies?
The random assembly of heavy and light chains.
86
Describe the structure of the T cell receptors
Made of two chains, alpha and beta. Has 4 regions:
1. Variable region
2. Constant region (embedded in cell membrane)
3. Joining region
4. Diversity region (only on beta chain, between variable and joining region)
87
How many V genes in alpha and beta chains of T cell receptors?
~70 in alpha, ~50-70 in beta
88
How many J genes in a alpha and beta chains of T cell receptors?
~61 in alpha, ~13 in beta
89
How many D genes in beta chain of T cell receptor?
2
90
What chain of T-cell receptors contain the diversity (D) region
Beta
91
What are 2 differences between T cell receptors and B cell receptors
Unlike immunoglobulins, TCRs are never secreted from the cell, and T-cell activation requires the presentation of a foreign peptide along with an MHC molecule, for TCR recognition (T-cells can't directly bind the antigens, unlike B cells)
92
Which of the following is necessary for B cell activation leading to antibody production?
a) B cells must first phagocytose the antigen and present it via Class I MHC
b) Helper T cells cytokines stimulate B cells that have independently recognized the antigen through BCRs (membrane-bound immunoglobulins)
c) B cells recognize antigens only through T cell help, without direct antigen binding
d) B cells release antibodies before differentiating into plasma cells
e) B cells require direct interaction with dendritic cells for activation
B
93
What is the length and location of the Major Histocompatibility Complex (MHC) genes?
Over 200 genes within a 4 Mb segment on chromosome 6's short arm
94
True or false: individuals with deletions on chromosome 6 tend to have severe immunodeficiencies
True
- due to lack of MHC genes
95
Class I MHCs bind to which type of T cells?
CD8+ T cells
96
Class II MHCs bind to which type of T cells?
CD4+ T cells
97
What 6 genes make up the class I region?
HLA-B, HLA-C, HLA-E, HLA-A, HLA-F, HLA-G
98
What 3 genes make up the class II region?
HLA-DP, HLA-DQ, HLA-DR
99
True or false: not much is known about the class III region
True
100
What causes the genetic diversity in MHCs among individuals?
Class I MHC loci (typically HLA-B, HLA-C, HLA-A) exhibit extensive allelic diversity, contributing to significant variability among individuals; it spans 1.8 Mb, encompassing numerous genes and pseudogenes
101
What is the role of class I MHCs?
Forms complexes with foreign peptides (similar to class II), and is crucial for cytotoxic T-cell recognition and response; essential for initiation cytotoxic T-cell responses against infected (virus peptides expressed in cell) or abnormal cells (mutated cells)
102
In general, how can pathogens evade the immune system?
Some viruses can escape detection by cytotoxic T cells by suppressing MHC class I gene expression in host cells (similar immune evasive processes have been found in cancerous cells), or can also suppress the genes that process the peptides and load them onto MHC molecules in APCs
103
What is the historical insight for the major histocompatibility complex?
MHC was discovered in the 1940s through mouse tissue graft experiments; differences in class I alleles led to graft rejection
- Conducted allele matching for transplant tolerance
104
TAP1 and TAP2 gene location and function?
Location: 6p21, located with MHC molecules
Function: transporter molecules that process foreign peptides and carry them to the ER (to be presented on MHC)
105
RAG1 and RAG2 gene location and function?
Location: 11p13
Function: Recombinases that participate in VDJ somatic recombination
106
What would happen to B cells and T cells if RAG1 and RAG2 genes were not present?
B cells and T cells would die if these recombination genes were not present
107
Define immunodeficiency diseases (IDs)
Occur due to absence or malfunctioning of any component(s) of the immune system (T cells, B cells, MHC, complement proteins)
108
Primary ID definition, # of syndromes identified
Genetic IDs; over 300 syndromes identified
109
Secondary ID definition
Result from external factors (radiation, infection, drugs), e.g. HIV/AIDS
110
Describe the effects of B-cell immunodeficiencies
Mainly affect B cells; absence of B cells leads to susceptibility to recurrent bacterial infections (due to a lack of memory B cells)
111
Describe the effects of T-cell immunodeficiencies
Affect T cells but can completely block B cell response: impact humoral response due to the dependency of B-cell proliferation on helper T cells
- Severe cases can lead to severe combined immune deficiency (SCID; for example, due to RAG1 and RAG2 deficiency, leading to B-cell and T-cell deficiency), makes patient susceptible to opportunistic infections; bone marrow transplants are critical.
112
True or false: SCID is one of the diseases tested for in newborns
True
113
What is the United States Immunodeficiency Network (USIDNET)?
Funded by NIH, a research consortium established to advance scientific research in the field of primary immunodeficiency (PI)
114
What is the genetic cause of X-linked SCID, and its effect on T cells, B cells, NK cells, TCRs and antibodies?
Genetic cause: IL2RG mutation (receptor for one of the cytokines released from helper T cells, IL2)
T cells: Absent
B cells: Non-functional
NK cells: Absent
Effect on TCRs and antibodies: No T cell activation, defective B cell response
115
What is the genetic cause of ADA-SCID, and its effect on T cells, B cells, NK cells, TCRs and antibodies?
Genetic cause: ADA (Adenosine deaminase) deficiency
T cells: Absent
B cells: Absent
NK cells: Absent
Effect on TCRs and antibodies: No TCR or antibody production
116
What is the genetic cause of RAG1/RAG2 SCID, and its effect on T cells, B cells, NK cells, TCRs and antibodies?
Genetic cause: RAG1/RAG2 mutation (defective V(D)J recombination)
T cells: Absent
B cells: Absent
NK cells: Present
Effect on TCRs and antibodies: TCR and BCR gene rearrangement fails
117
What is the genetic cause of JAK3 SCID, and its effect on T cells, B cells, NK cells, TCRs and antibodies?
Genetic cause: JAK3 mutation (affects cytokine signaling)
T cells: Absent
B cells: Non-functional
NK cells: Absent
Effects on TCRs and antibodies: No functional TCRs, defective B cell response
118
Is cancer an immunodeficiency disease?
It depends on how you define it, but generally yes. Detection of mutated cells by T cells should get rid of tumour cells. These mutations happen all the time but do't lead to cancer due to a functional immune system
119
How would cancer immunotherapies differ from traditional cancer therapies?
Traditional cancer therapies, like radiation or drugs, kill both healthy and cancerous cells (as its the general proliferation of cells that is targeted by traditional cancer therapy.
- We can train the immune system to look for certain oncogenic mutations, so that it specifically targets only cancerous cells
120
What are two GENERAL components of Thymus selection of T cells?
1. Effectiveness
- Each clone should be able to recognize and mount a response against a foreign antigen
2. Safety
- Have to make sure that they don't start detecting your own organs/tissues
121
What is positive selection for T cells in the thymus?
All the T cells that have the ability to bind to MHCs/antigens (self and non-self) are selected for.
122
What happens after positive selection for T cells in the thymus?
Negative selection: T cells with reactivity for foreign antigens are activated during infection
123
Where are T cells deposited after negative selection?
Into the lymph nodes
124
True or false: Positive and negative selection happens in the same region of the thymus
False; positive and negative selection happens in different regions of the thymus
125
True or false: mutations that result in cancer generally arise in somatic stem cells
True
126
How do mutant cells specifically evade the immune system to initiate cancer?
They escape T cells (tumour-specific T cells are dysfunctional)
127
How is cytotoxicity from the immune response suppressed/modulated?
Additional molecules are engaged, such as PD-1 (in phase 1 and 2, early and late dysfunction, respectively) that act as an "immune checkpoint" and stop the T cell response.
Molecules other than PD-1are also expressed in phase 2 , CD38, CD39, CD101, TIM3 which promote tumour growth rather than acting as a suppressor
128
What molecule(s) act as immune suppression proteins in phase 1 (early dysfunction) of tumour growth?
PD-1
129
What molecule(s) act as immune suppression in phase 2 (late dysfunction) of tumour growth?
CD38, CD39, CD101, TIM3
130
What happens to the immune system as a tumour expands (in general)?
The host starts expressing immune response suppressing molecules
131
How can we re-activate the immune system as a cancer treatment?
Creating neutralizing antibodies against immune checkpoints
- e.g. neutralizing the effect of PD-1, which can activate the immune system.
- One of the very first ways of modulating the immune system and creating immunotherapies
132
Other than neutralizing PD-1, what are 3 other methods of immunotherapy for cancer?
1. Inject immune cells with enhanced ability to secrete IL-2 cytokines.
2. Design T-cells and T-cell receptors to target the cancerous tumour
3. Design therapies to remove myeloid cells (myeloid cells express immune checkpoints)
133
Define populations
Local groups of people sharing a common gene pool
134
Define gene pool
The set of genetic information (e.g. alleles of a gene) carried by the members of a sexually reproducing population
135
Define population genetics
The study of genetic variation and how genes and genotypes are maintained or change in populations, NOT individual matings
136
How can populations be described? (4)
1. Age structure
2. Geography
3. Birth and death rates
4. Allele frequencies
137
True or false: individuals are more diverse than populations
- Give an example explaining why or why not
False; populations are more diverse than individuals
- e.g. only a group can carry all the alleles for traits such as blood types A, B, AB, and O
138
What are 5 reasons for why we study population genetics?
1. Identification of genetic risk factors
2. Enhancement of preventive healthcare
3. Promotion of health equity
4. Optimization of resource allocation
5. Advancement of personalized medicine
To achieve these objectives, alleles and their frequencies must be identified
139
Describe how studying population genetics helps with identification of genetic risk factors
Studying genetic variations within populations helps identify individuals at higher risk for certain diseases, enabling early interventions and treatment plans
140
Describe how studying population genetics helps with enhancement of preventive healthcare
Genetic screening programs can detect predispositions to diseases, allowing for timely preventive measures that reduce morbidity and mortality rates
141
Describe how studying population genetics helps with promotion of health equity
Incorporating diverse genetic data ensures that all population groups benefit from medical advancements, addressing health disparities and improving outcomes across communities
142
Describe how studying population genetics helps with optimization of resource allocation
Insights from population genetics guide healthcare systems in allocating resources effectively, tailoring services to the specific genetic profiles and health needs of different communities
143
Describe how studying population genetics helps with advancement of personalized medicine
Understanding genetic diversity facilitates the development of targeted therapies and interventions, leading to more effective and efficient healthcare solutions (able to determine if diseases in a population will affect individuals uniquely, requiring personalized care)
144
True or false: the frequency of alleles in a population may change from generation to generation, and changes in allele frequency can cause change in genotype frequency
True
145
True or false: we can always measure allele frequencies in populations by directly counting them
False; in cases of incomplete or co-dominance, we can count the alleles directly, but in cases of complete dominance, we need to use Hardy-Weinberg to estimate the allele frequencies (there's no direct link between genotype and phenotype in complete dominance because heterozygotes and homozygous dominant genotypes have the same phenotypes)
146
What specifically causes incomplete dominance?
Haploinsufficiency (one dominant allele is not enough to perform the job of 2 dominant alleles)
147
What specifically causes co-dominance?
Both alleles perform a normal function (both "dominant"), but expressed peptides are different between alleles
148
What is an example of codominant alleles?
The MN blood group
- M and N alleles are co-dominant and produce three phenotypes, M, N, and MN
149
What gene codes for the MN blood type and how can we directly measure allele frequencies with this specific example?
Glycophorin A encodes the red blood cell membrane protein
- 2 variants of protein can be easily detected with antibody testing in individuals
150
True or false: Hardy and Weinberg independently developed a mathematical formula to determine frequency of alleles when one or more alleles are recessive
True
151
What happens to allele and genotype frequencies in an "ideal" population from generation to generation?
Allele and genotype frequencies remain constant from generation to generation in an "ideal" population (free from many complications that affect real populations)
152
What are the 7 assumptions of the Hardy-Weinberg law?
1. The population is large enough that there are no sampling errors in measuring allele frequencies
2. All genotypes are equally able to reproduce
3. Mating in the population is random
4. No migration into or out of the populations (because different populations have different gene pools)
5. No new mutations are occurring
6. No matings between different generations
7. All matings produce the same # of offspring who are equally fertile
153
In the hardy weinberg equlibrium equation, why is it 2pq and not just pq?
Because there are two parents so 2 ways to be heterozygous
154
Define genetic equilibrium
When the allele and genotype frequency for a particular gene remains constant from generation to generation
- Equilibrium in a population explains why dominant alleles do not replace recessive alleles
155
True or false: when not in equilibrium, allele frequencies may stay the same between generations, however, the combinations of genotype frequencies could still change resulting in increased observed phenotype frequencies
True
156
What 3 things could the Hardy-Weinberg law be used to do?
1. Estimate frequencies of autosomal dominant and recessive alleles in a population
2. Detect when allele frequencies are shifting in a population (evolutionary change)
3. Measure the frequency of heterozygous carriers of deleterious recessive alleles in a population
157
For a genetic disorder inherited as a recessive trait, how are most disease-causing alleles carried? How is the risk of having an affected child calculated?
By heterozygotes
- The risk of having an affected child is calculated by using the frequency of heterozygous carriers of deleterious recessive alleles in a population
158
True or false: you can count recessive alleles in males for X-linked traits
True; the number of males with the mutant phenotype equals the allele frequency for the X-linked recessive trait
159
What is the frequency of an X-linked trait in males and in females? When would this not hold true?
In males: q
In females: q^2
This would not hold true if you have more than 2 alleles for a gene
160
In ABO blood types, six different genotypes are possible. What is the expanded Hardy-Weinberg equation for genotype frequencies? For allele frequencies?
Allele frequencies: p(A) + q(B) + r(O) = 1
Genotype frequencies: p^2 (AA) + 2pq (AB) + 2pr (AO) + q^2 (BB) + 2qe (BO) + r^2 (OO) = 1
161
True or false: estimating the frequency of heterozygotes in a population is an important part of genetic counselling
True
162
We share ____% of our DNA, and are different at _____ bases
99.9
3,300,000
163
What are the 3 levels of genetic variation in normal populations?
1. Chromosomal level
2. Sub-chromosomal level (copy number variation
3. Single nucleotide level
164
DiGeorge syndrome is caused by...
A partial chromosome imbalance (22q11.2 del)
165
Achondroplasia is caused by...
A single nucleotide substitution (Gly380Arg in FGFR3 gene)
166
What are 4 things that determine benign from disease-causing variants?
1. What type of variation?
2. Where in the genome is it? (i.e. in the middle of a gene or not)
3. What is the gene(s) involved? (some sequences are tolerant to mutations)
4. What is the variant effect? (i.e. pathogenic, benign, unknown)
167
What 3 terms are no longer used?
SNP, polymorphism, mutation
168
Define common variant
>1% allele frequency in a population
169
Define rare variant
<1% allele frequecny in a population
170
What are 4 examples of small variations?
1. Single nucleotide variant (SNV)
2. Small nucleotide insertion/deletion
3. Dynamic repeat
4. Microsatellite
171
Define copy number variation (CNV)
Deletion or duplication of one or more exons
172
What is the most common type of sequence variation?
Single nucleotide variants (SNV)
173
Number of SNVs per person and effects?
>3,000,000
Variable effects: benign to disease-causing
174
What could a variant in the promoter of a gene likely result in?
Transcription would likely be affected, resulting in altered gene expression
175
What would happen if the first nucleotide of the pre-mRNA is altered? Last nucleotide?
First nucleotide: can't add 7mG cap which leads to degradation of pre-mRNA, resulting in decreased expression
Last nucleotide: same thing but can't add poly A tail
176
How do you number exons?
Nucleotide #1 = A of "ATG"
- upstream nucleotides are negative (e.g. 2 bp upstream = -2)
- downstream coding nucleotides are numbered consecutively (omit the introns)
177
If the 90th nucleotide in a coding region of a gene is A, how would you write this?
c.90A
178
If the 90th nucleotide in a coding region of a gene was A but changed to G, how would you write this?
c.90A>G
179
How do you number intronic sequences?
Numbered according to the first or last coding nucleotide in the nearest exon and the number of nucleotides downstream from the splice donor site (+) or upstream from the splice acceptor site (-)
- Changes halfway through the intron
180
If the 5th intron nucleotide downstream from the 90th nucleotide in a coding region of a gene changes from G to C, how would you write this?
c.90+5G>C
181
How are amino acids numbered in a protein sequence?
Amino acids are numbered consecutively starting with the initiation codon = 1
182
If the 31st amino acid in a protein sequence is phenylalanine, how would you write this?
p.Phe31
183
How do you write a synonymous (silent) variant?
e.g. for CTA to CTG at 325th amino acid (both code for Leu), write p.Leu325=
= means that amino acid doesn't change
184
How do you write a non-synonymous (missense) variant?
e.g. for CTG to CCG at 112th amino acid (Leu to Pro), write p.Leu112Pro
185
How do you write a nonsense variant?
e.g. for TTA to TAA at 33rd amino acid (Leu to Stop), write p.Leu33Ter or p.Leu33*
186
Truncated proteins can be detected by the cell through what process?
Nonsense mediated decay
187
Describe nonsense mediated decay
Pre-mRNA recruits all of the splicing proteins/rRNAs. The ribosome loads onto it and starts sliding along mRNA to find the start codon. As it scans, it bumps the proteins and rRNAs off. Once it starts translating, it also bumps off the proteins and rRNAs. If you have a premature stop codon, there's going to be splicing proteins remaining on the mRNA that shouldn't be there. This signals to the cell that this mRNA has a nonsense variant in it.
188
What is a read-through variant?
DNA sequence change results in a stop codon loss
189
If you had a stop codon at amino acid 807 switched to Glycine, how would you write this?
p.Ter807GlyextTer102 (the normal stop codon is now a glycine, and it has been extended to the next stop codon which is 102 amino acids downstream of the Gly)
190
True or false: DNA sequence changes at the +1, +2, -2 and -1 positions almost always affect splicing
True
191
What 3 things could happen when a splice site is disrupted?
1. Spliceosome can search for a cryptic donor site (upstream or downstream GU that acts as the donor site instead)
2. Intron inclusion
3. Exon skipping (could affect multiple exons)
192
What is the nucleotide nomenclature for frameshift mutations? Ex: use nucleotide at position 1147 deleting a C, resulting in His changing to Met and 383 and the next stop codon being 75 codons downstream
c.1147delC
p.His383MetfsextTer75
193
What is the amino acid nomenclature for frameshift mutations? Ex: use His at position 383 changing to a Met, resulting in a frameshift that encounters a stop codon 75 amino acids downstream
p.His383MetfsTer75
194
Main difference between germline and somatic variant phenotypes?
With somatic variants, phenotypes are only seen in tissues with the variant
195
Where do most of sequence variations come from?
Replication errors
196
What is the replication error rate for cells in units of errors/base/cell division? What about error/division?
10^-10 errors/base/cell division
1 error/1.5 divisions
197
How many bases per cell per day are damaged?
10,000-1,000,000
198
What two things can cause DNA damage?
1. Natural processes (deamination, depurination, UV light)
2. Mutagens
199
What is deamination and provide an example
The removal of an amino group from a nucleotide resulting in a sequence variant
e.g. 5-Methylcytosine deaminating into Thymine
200
What is depurination?
The ribose backbone stays, but the rest of the nucleotide falls off. So then during DNA replication, the polymerase will come along, find an empty nucleotide, and just add whatever it wants to it.
201
If you see male to male transmission on a pedigree, what form of inheritance does this rule out?
X-linked
202
What 3 things are seen in X-linked recessive pedigrees?
1. Only affects males
2. Affected males have normal daughters
3. No male to male transmission
203
True or false: Fragile X syndrome doesn't really fit any of the inheritance patterns seen in pedigrees
True
204
What is a dynamic repeat and what are they a major cause of?
Expansion of a simple repeat in a coding region or non-coding region
Neurological disorders
205
Dynamic repeats often demonstrate which two things?
Reduced-penetrance alleles and variable expressivity
206
What is reduced-penetrance alleles?
a gene variant (allele) where not everyone who inherits it will develop the associated trait or disease
- e.g. some people with fragile X syndrome develop a phenotype and others don't, depending on the length of the repeat.
207
What are 2 potential consequences of expansion?
1. Loss of function
2. Novel function
208
What are two examples of loss of function expansion alleles?
Fragile X (X-linked recessive), Friedreich Ataxia (intronic) (Autosomal recessive)
209
What are two examples of novel function expansion alleles?
Huntington (coding), Myotonic Dystrophy (3'UTR) (Autosomal Dominant)
210
What causes fragile X syndrome?
A repeated(CGG)n element near the promoter of the FMR1 gene
211
What is the normal number of CGG repeats?
6-54
212
What is the premutation number of CGG repeats? What could this result in?
55-200
- You may develop an adult onset of the disease later in life, but you are at risk of having a kid with Fragile X because it expands in your germ line
213
What is the full expansion number of CGG repeats
200-1000's
214
What is the mechanism of disease for Fragile X?
Expansion of the CGG repeat causes methylation of the repeat and because it's in the promoter, it shuts down the fMR1 gene which is involved in neural development
215
Why doesn't fragile X end human civilization?
Because people with fragile X tend not to reproduce
216
It's always the (male/female) carriers of the CGG repeat that expands
Female; expansion only happens in the maternal lineage
217
The risk of expansion is dependent on what?
The pre-mutation size
218
A premutation carrier female (will/will not) have affected offspring
Will
219
A premutation carrier male (will/will not) have affected offspring
Will not
- Will have premutation carrier offspring an affected grandsons (from his daughter)
220
What is anticipation?
Earlier age at onset through successive generations of a pedigree
221
True or false: the size of the repeat expansion correlates with the age of onset
- Explain why or why not
True; the larger the expansion, the earlier the age of onset
222
What type of repeat is myotonic dystrophy type I?
CTG repeat in the 3'UTR of the DMPK gene
223
How many repeats in CTG repeat in mild DM1?
50-150
224
How many repeats in classic DM1?
100-1000
225
How many repeats in congenital DM1?
>1000
226
What is the protein nomenclature for start codon variants?
p.Met1?
(no translation so can't incorporate the amino acid that it changes to)
227
What is a microsatellite?
Repeat units of 2-5 nucleotides in length that are also called short tandem repeats (STR), most of which do not have any clinical impact.
228
Microsatellites are also known as...
Short tandem repeats (STR)
229
Microsatellites are ________
- Explain what this means
Polymorphic
- >10,000 known across the genome
230
True or false: There's often many alleles (i.e. number of repeat lengths) in normal populations
True
231
True or false: microsatellites cannot be detected with PCR
False; they're easily detected using PCR
232
What 3 things can microsatellite amplification via PCR be used for?
1. DNA fingerprinting
2. Relatedness (e.g. twin studies, paternity)
3. Disease-gene genomic localization (finding where disease genes are in the genome)
233
Describe how microsatellites can be used for paternity testing
Can see if one of two microsatellites (cause one per chromosome) matches that of the father
234
Describe how microsatellites can be used for forensics
They use 15 microsatellites scattered across the genome
- There's a 1 in 5 billion chance that 2 people have the same microsatellites
235
Describe how microsatellites can detect chromosome aneuploidies
- use Down's Syndrome as an example
People with Down's syndrome have extra set of microsatellites on chromosome 21 which can be detected via PCR
236
What causes large deletion/duplication variants (Copy number variation)?
Faulty alignment of homologous chromosomes which leads to duplication and deletion during recombination
- Can be millions of bps long
237
What type of variation is Charcot-Marie-Tooth (CMT1a) caused by?
Duplication of PMP22 gene
238
What type of mutation is Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) caused by?
Deletion of PMP22 gene
239
Describe Factor VIII inversions and what disease is caused by them
Causes haemophilia A
- Certain introns are facing each other, and recombination allows for inversion, which basically breaks a gene in half
240
What is the most common cause of X-linked Duchenne muscular dystrophy?
Exon-level duplications/deletions of the DMD gene, where one or more exons are duplicated (rare) or deleted
241
A third of all X-linked Duchenne muscular dystrophy patients have a de novo event (i.e. not found in mom). What predisposes patients to this high rate?
Alu-repeat-mediated recurrent CNVs
- The introns of the DMD gene are littered with Alu repeats which are highly similar and predisposes patients to homologous recombination (resulting in exon duplications/deletions)
242
Loss of function variants tend to be (dominant/recessive)
Recessive
243
What is the difference between loss of function variants and haploinsufficiency?
Both copies are affected in loss of function
244
Why are 1/25 individuals of European descent CF carriers?
Due to the heterozygote advantage. Retaining bodily fluids was advantageous when cholera was prominent
245
What does the clinical severity of loss of function variants correlate with?
Residual activity
246
Gain of function variants correlate with (dominant/recessive) disorders
Dominant
247
Achondroplasia is an example of a (GOF/LOF) variation
GOF
248
Congenital adrenal hyperplasia is an example of a (GOF/LOF) variation
LOF
249
Why is the sickle cell anemia variant have a higher prevalence in African populations?
Due do heterozygote advantage of being a sickle cell anemia carrier against malaria
250
What is a dominant negative variant?
A variant that results in a protein that adversely affects the normal product within the same cell (1 normal copy and variant in other copy)
- This means that even if you still have one working (normal) copy of the gene, the mutated copy can still cause disease or dysfunction.
251
What type of variant is brittle bone disease (osteogenesis imperfecta)?
Dominant negative variant
252
Describe the mechanism for how osteogenesis imperfecta results in its phenotype
Gly in collagen gene is substituted for another amino acid so the collagen chains can't pack together and form a trimer
253
True or false: incorporation of abnormal collagen in osteogenesis imperfecta is more detrimental and results in a more severe phenotype
True
254
What is a heterochronic/ectopic expression?
Are they usually dominant or recessive disorders?
Gene is expressed at the wrong time (heterochronic) or in the wrong place (ectopic)
- Usually dominant
255
What type of disorder is lactose tolerance?
Heterochronic expression because lactase is supposed to only be expressed in childhood but with lactose tolerance it's expressed in adulthood as well
256
Haploinsufficiency is inherited as (dominant/recessive) disorders
Dominant
257
True or false: haploinsufficiency is only generated de novo
False; they can be inherited or de novo variants
258
True or false: haploinsufficiency is often expressed with variable expressivity
True
259
What are gene dosage diseases?
Extra copies of normal gene products that are sufficient to cause disease
260
Gene dosage diseases are inherited as (dominant/recessive)
Dominant
261
Gene dosage disorders may have variable....
Penetrance and expressivity
262
What is an example of a gene dosage disorder that we discussed?
Charcot-Marie Tooth Disease
- Caused by extra copy of PMP22 gene
263
True or false: Genes can have both loss and gain of function variants
True
264
Mendel's law of segregation
Every individual has a pair of alleles for each trait from each parent and will randomly pass on one to their offspring
265
Mendel's law of independent assortment
- Describe what you would see with two markers if unlinked according to this law
Separate genes for separate traits are passed independently to offspring
- If unlinked, alleles from marker 1 will be found equally associated with both marker 2 alleles
266
What is the exception to Mendel's law of independent assortment?
- Describe what you would see for two markers in this exception
Linkage
- If linked, one allele from marker 1 will be found associated with one allele from marker 2 > 50% of the time
267
Define genetic linkage
The tendency of characters (phenotypes or marker alleles) to co-segregate in a pedigree because they lie in close proximity on a chromosome
268
Define genetic marker
DNA sequence that has classifiable alleles that allow the marker to be tracked through families (i.e. can tell the chromosomes apart, know which one came from mom and which one came from dad)
269
Difference between uninformative and informative markers?
Uninformative marker: can't tell which parent each allele came from
Informative: can tell which parent each allele came from
270
What does phase refer to in genetic linkage analysis?
the combination of alleles inherited from one parent.
271
What score is calculated to know the probability of linkage?
The LOD score (Z)
272
What does a LOD score >3 indicate?
1000:1 odds in favour of linkage (linkage confirmed)
273
What does a LOD score of <-2 mean?
100:1 odds against linkage (linkage disproven)
274
True or false: The LOD scores from linkage analysis of multiple families can be added together to reach a LOD score of >3
True
275
1% recombination frequency equals to how many centimorgans?
1 cM
276
What did genetic maps made before the human genome was even published allow for? And how was this done?
This was done by ordering the map of markers (mainly microsatellites) generated by genotyping large families
- Distance between markers is the recombination rate
- Provides a framework for linkage mapping
277
True or false: Genetic mapping allowed for the development of paternity testing
True
278
Describe what you would see (according to the law of independent assortment) if you had a disease allele and a marker allele unlinked
If unlinked, disease state will be found equally associated with both marker alleles
279
Describe what you would see (according to the law of independent assortment) if you had a disease allele and a marker allele linked
If linked, disease state is found associated with one allele >50% of the time
280
Define positional cloning
One of the uses of genetic linkage, identifying the genomic location of a disease gene without any prior knowledge on where or what the causative gene is
281
True or false: double recombinants are common in nature
False; double recombination rarely occurs due to interference (when recombination at one locus prevents recombination at other spots)
282
What would be the probability of developing a disease linked to 2 markers if you inherit both markers?
100% because double recombination doesn't exist in nature
283
What is the relationship between apriori knowledge and amount of genome that you have to sequence?
If you know what you're looking for, you can be very selective in what you sequence. However, if you don't know what you're looking for, you have to sequence a lot of the genome
284
What is the relationship between apriori knowledge and number of variants that you have to sequence?
If you know what you're looking for, you can be very selective in what you sequence. However, if you don't know what you're looking for, the number of variants that you find will increase dramatically.
285
In Rett syndrome, 88% of girls have a heterozygous variant in the MECP2 gene on the X chromosome. Males with classic Rett syndrome are exceedingly rare. There is no family history of any developmental delay. What is the mode of inheritance?
X-linked dominant (de novo)
- Dominant bc the phenotype shows in heterozygotes
- de novo bc there's no family history of delay
286
Why are males with classic Rett syndrome exceedingly rare?
Because males only have one X chromosome so having the copy mutated MECP2 gene is lethal (don't have another X chromosome to partially compensate)
287
How would you identify Rett syndrome?
Using dideoxy sequencing (PCR amplify and Sanger sequence the coding region)
288
What type of variants is Sanger sequencing able to identify? Which does it not identify?
Able to detect missense, frameshift and splicing variants
Unable to detect deletions and duplications
289
The MECP2 sequencing results for Rett syndrome shows two heterozygous variants, one being missense and one being nonsense. What is the most likely/more common variant if LOF variants are the pathogenic mechanism in Rett syndrome?
The nonsense variant because they are more likely to be LOF (nonsense variants are more damaging)
290
A pedigree for Leigh disease shows generation skipping and multiple people affected in one generation with unaffected parents. The parents are also third cousins. What is the most likely mode of inheritance in this family?
Autosomal recessive
291
How can we identify where to start looking for identifying the genetic cause of Leigh disease (disease caused by inbreeding)
Homozygosity (or identity by descent) mapping
292
Describe homozygosity (or identify by descent) mapping
Assumption: The variant was introduced into the population by one ancestor
- each individual carries the same variant on both chromosomes (homozygous for the variant) and all affected individuals carry the same variant.
The amount of identity to the original chromosome diminishes with each succeeding generation (the region around the "variant" will be identical on each chromosome, there's starting to be recombinations that are starting to reduce the window that's identical between these two chromosomes).
Researchers look for DNA regions that are the same in sick people but different in healthy people which helps them find the mutation without knowing where to look beforehand (look for regions where all affected are homozygous for the same allele but none of their unaffected sibs are
293
Explain how homozygosity (or identify by descent) mapping is a type of positional cloning
It determines the location of a causative gene without previous knowledge of it's location in the genome or gene function (hypothesis-free approach)
294
What is IBD mapping and what are the two ways that you can go about identifying regions of identity by descent?
IBD mapping: "Identity-by-Descent mapping," is a genetic analysis technique that identifies regions of the genome where individuals share DNA segments inherited from a common ancestor.
1. Not common anymore: Microsatellite analysis helps detect regions of identity by descent (IBD) by checking if affected individuals have the same microsatellite sequences on both chromosomes, meaning they inherited them from a common ancestor (if there's an LOD >3 with the IBD region and a disease gene, this confirms that the IBD region is actually linked to a disease)
2. Take a DNA sample and genotype multiple SNVs at a time to look for individuals that are homozygous for different alleles
295
What is a caution for IBD mapping using SNVs to find homozygosity across the genome?
All of us are homozygous in 1-5% of our genome.
296
What's the difference between identity by descent and identity by state?
Identity by descent: Individuals share the same alleles that was originally derived from a common ancestor
Identity by state: Both parents are homozygous for an allele so they have no choice but to be homozygous for the same allele
297
Describe what the B allele frequency would look like on chromosomes that is identical by descent
Most regions have all three genotypes but then some regions will have missing AB genotype
- Middle region is blank because it's the centromere (no SNVs in this region that you can genotype)
- Really heterozygous near the end because in every meiosis I, there is at least one crossover between the p arms and one between the q arms (that's what synapses the chromosomes together)
298
4 chromosomal regions were found to have linkage with the Leigh disease phenotype. How did they determine exactly which gene was causing the disease?
They used a database so they knew that one of the regions had a gene that coded for an assembly factor for complex I, so they did Sanger sequencing on it and found a homozygous frameshift variant in this gene
299
Why is gene hunting not easy?
Gene identification can be laborious and expensive using Sanger sequencing
- Candidate regions can be large with lots of genes, no good candidates
300
Kabuki syndrome is characterized by very rare familial recurrence (sib-sib and parent-child), and males are equally affected as females. Individuals with Kabuki syndrome also don't reproduce. What are the possible modes of inheritance?
De novo dominant (not inherited from paraents)
301
True or false: you can identify the molecular cause for Kabuki syndrome using linkage
False, because Kabuki syndrome isn't inherited
302
What method do you need to use to identify the molecular cause for Kabuki syndrome, since you can't use linkage? Describe the steps (5)
Next-Generation sequencing (aka Massively Parallel Sequencing)
1. Take the genomic DNA, break it up into a bunch of little bits, add some tags of these little bits
2. Allow bits to be fixed to a solid surface (a slide or a bead).
3. Wash the nucleotides over the surface
4. Have a detector that will read the incorporation of these nucleotides
5. Map each sequence (read) back to the reference genome using bioinformatics (has problems)
303
How are you able to get a consensus sequence for a sample that you've sequenced using NGS?
It's by piling up reads that you're able to get the consensus sequence for the sample that you've sequenced
304
What is the depth of coverage when using NGS?
How many times did you independently sequence each region of the genome?
305
Compare dideoxy sequencing with NGS in terms of speed
dideoxy sequencing is way faster (days) compared to NGL (1 week)
306
Dideoxy sequencing is position-(dependent/independent) while NGS is position-(dependent/independent)
Dependent (know where the sequence came from because it should be between your primers), independent (have to figure out where the sequence came from)
307
Which method is more accurate: dideoxy sequencing or NGS?
Dideoxy
308
Which method requires bioinformatics support: dideoxy or NGS?
NGS
309
What is exome sequencing?
Sequencing only protein coding exons (which comprises 2% of the genome)
310
What percentage of Mendelian variants are estimated to be in exons?
~80%
311
What are 4 limitations of exome sequencing?
1. Not all disease variants are coding
2. Not all exons are captured
3. Exon level deletions/duplications are hard to detect
4. Inversions and dynamic repeat expansions are not detected
312
What is the range for # of variants/exome (i.e. how many sequence changes are present in a patient compared to the reference genome? Why such a big range?
20,000-40,000
Big range is due to 70% of the reference genome being made up from 1 white guy
313
How do you dramatically reduce the search space to identify the sequence change in Kabuki syndrome for a de novo dominant variation?
Find all variants present in child but not in parents and compare them, which dramatically reduces search space
314
How do you identify the sequence change in Kabuki syndrome for multiple patients with the same phenotype (dominant or recessive)?
Take a bunch of people that you think all have the same rare genetic disease find all of the genes that they each have variants in. Then see where thevariants overlap.
315
How do you identify the variant in recessive disorders with consanguineous parents?
Look for a homozygous rare variants that show identity by descent, which narrows down the mapping region.
i.e. 20,000 variants -> 3,000 rare variants -> 3 in IBD region
316
How can we use pedigrees to determine the variants present in large autosomal dominant or x-linked genes?
Sequence the most distantly related people with the disease and compare which variants they have that are similar (most distant relatives used to reduce the number of shared variants that are unrelated to the disease)
317
How can you identify somatic (mosaic) variants?
Sequenced affected tissue and unaffected tissue and compare them
318
Proteus syndrome affected gene and protein sequence change
AKT1 gene, p.Glu17Lys
319
What is the first step in looking for the variant that causes a disease?
Look at variants in normal populations and see which variants are rare or common. If the disease is rare (<1% allele frequency in population), you can filter out the rare variants and then keep filtering based on if it's found in the coding region, if they're nonsense, nonsynonymous, etc.
320
Why are there so many non-functional variants in a population? (65 variants)
Because these individuals are usually heterozygous for the variant, so the other gene compensates for any loss
321
What are 3 considerations in variant classification?
1. Population prevalence of the variants
2. Whether the genotypes have been previously associated with disease (including mode of inheritance of the disease)
3. SNV classification if you find SNVs (i.e. what is the predicted effect? what is the computer-based prediction model of that effect? etc)
322
Whole genome sequencing produces (short/long) reads
Short
323
What 5 things is whole genome sequencing able to detect?
SNVs, CNVs, dynamic repeat expansions, intronic variants, and GC rich reginos
324
What are 3 limitations with whole genome sequencing?
1. Variant interpretation is challenging (there's ~5,000,000 variants/trio, trio-exome sequencing analyses genetic data from 3 people, including the baby, its mother, and the father)
2. Instrument capacity
3. Data storage
325
What are the 2 different methods of long read genome sequencing?
1. Pacific Biosciences SMRT sequencing
2. Oxford nanopore technologies sequencing
326
Describe Pacific Biosciences SMRT sequencing (method of long read genome sequencing)
Create little wells, and then put ssDNA into them with DNA polymerase attached to the bottom of the slide. The DNA polymerase adds dNTPs that are fluorescently tagged, which are detected by a camera at the bottom of the well
327
Describe Oxford Nanopore Technologies sequencing
Nanopore has current running through it and as ssDNA passes through nanopore, current is disrupted. Each nucleotide has a characteristic disruption.
328
What are 3 benefits of long read genome sequencing?
1. Very long reads (15,000 to 4,000,000 bp)
2. Increased detection of structural variants, repeat expansions
3. Native DNA used - reads DNA methylation
329
What are 3 disadvantages of long read genome sequencing?
1. $$
2. Decreased throughput
3. Decreased accuracy
330
What is the primary advantage of exome sequencing over whole genome sequencing?
Exome sequencing focuses on 2% of the genome where approximately 80% of disease-causing variants are found
331
True or false: mitochondria are only important for generation of ATP via oxidative phosphorylation
False; they're also important for many other processes, such as apoptosis, cell signalling, metabolism, ROS, immunity, Ca2+ homeostasis, iron-sulphur biosynthesis and steroid production
332
Describe endosymbiosis theory in general
Mitochondria were a free-living bacteria taken up by a eukaryotic cell. Both mitochondria depends on host and host depends on mitochondria (not just on its energy, but metabolic pathways as well)
333
What 4 things provide evidence for endosymbiosis theory?
1. mtDNA (mitochondria have their own genome like bacteria)
2. Dedicated machinery for transcription and regulation
3. Have their own ribosomes
4. Lipids (cardiolipin is a lipid found in eukaryotic cells only within the mitochondrial membrane and bacterial membrane)
334
True or false: the mitochondrial DNA codes for 13 proteins, so the mitochondrial proteome is 13 proteins
False; mtDNA does code for only 13 proteins but many proteins in the mitochondria are actually produced in the nucleus and transported to the mitochondria
335
Describe the mitochondrial genome structure
Basically a large plasmid with very compact structure (very little non-coding DNA), except for the D loop
336
Describe the mitochondrial DNA organization in the mitochondria
Organized in a nucleoid structure and packaged by a protein called mtTFA/TFAM (like histones for mtDNA)
337
What is the expression and replication of mtDNA regulated by?
Nuclear-encoded proteins
338
What is the number of protein-coding genes, tRNA genes and rRNA genes in mtDNA?
Protein-coding: 13
tRNA: 22
rRNA: 2
339
True or false: All mtDNA proteins are involved in oxidative phosphorylation and are localized within the inner membrane of the mitochondria
True
340
mtDNA is (linear/circular)
Circular
341
mtDNA is ____ bases long, while nuclear DNA is ____ bases long
mtDNA: 16.5 kb
nuclear DNA: 3.3 billion
342
True or false: codon usage differs between mtDNA and nuclear DNA of the same species
True
343
mtDNA mRNA is (monocistronic/polycistronic)
Polycistronic
344
mtDNA has a (lower/higher) mutation rate than that of nuclear DNA
- Explain why
Higher (10X higher)
- Has a lower repair rate (no homologous recombination and lots of ROS made in mitochondria that causes damage to bases)
345
Start codons in mtDNA
AUA and AUG
346
Stop codons in mtDNA
AGA and AGG (UGA codes for Trp)
347
What happens to mtDNA upon binding of mtTFA/TFAM?
It forms a U-shaped bend in DNA, so as you increase the ratio of TFAM to DNA, this increases the folding of mtDNA
348
What 3 proteins are required for mtDNA replication?
1. Polymerase gamma (A and B subunits, form a dimer)
2. Helicase (TWINKLE)
3. mtSSB (Single stranded binding protein)
349
Describe the D-loop in mtDNA
Forms a triple helix DNA structure, contains 3 promoters for transcription: HSP1, HSP2, LSP
350
HSP1 promoter transcribes which genes?
3 tRNAs and 2 rRNAs, then terminates
351
HSP2 promoter transcribes what?
A long transcript that goes all the way around the circular mtDNA genome
352
LSP promoter transcribes what?
A long transcript that goes all the way around the circular mtDNA genome (opposite direction of HSP2)
353
How are large amounts of ribosomes made in the mtDNA?
HSP1 promoter and 2 rRNA genes (12S and 16S rRNA) forms loop and transcription machinery happens with this loop over and over again
354
How many protein coding genes on mtDNA heavy strand?
12
355
How many protein coding genes on mtDNA light strand?
1
356
Describe the tRNA punctuation model
tRNA genes tend to be interspersed intermittently with all of the mRNA open reading frames. This interspersion allows for tRNAs to mature by cleave at their 5' and 3' ends while releasing mRNAs and rRNAs and the same time
357
Has epigenetics (methylation) been observed in mtDNA?
Some methylation has been reported and has been correlated with disease, but no mechanism (don't know if the methylation is causing increased or decreased transcription). But there's also evidence that this could be an artifact of the methods we use to look at DNA methylation.
358
Has epigenetics (phosphorylation/acetylation) been observed in mtDNA?
TFAM has been shown to be phosphorylated and acetylated, but we haven't figured out the functional consequences of these modifications.
359
True or false: the mitochondria can influence nuclear epigenetics, and the nuclear epigenetics can influence mitochondrial epigenetics
True
360
Describe how mtDNA replication and fission are linked
mtDNA in mitochondria is synthesized and the ER initiates a fission event between the two mitochondrial genomes. Afterwards, you end up with two separate mitochondrial species and each one of them has a copy of the genome.
361
Describe how mtDNA replication and fusion are linked
Proteins such as MFN1, MFN2 and OPA are involved in fusing the mitochondria network together. When these proteins aren't function, the mitochondrial network is more broken down into a bunch of small pieces that aren't properly distributed for mitochondrial division.
362
True or false: there needs to be a balance between fusion and fission for proper mtDNA distribution during mitochondrial division
True
363
Mitochondria appears more elongated during which phase of the cell cycle?
G1-S
- Makes sense bc mtDNA is replicating
364
Mitochondria appears more fragmented during which phase of the cell cycle?
G2 and M (ensures that you get an even segregation of mitochondria to both of the daughter cells)
365
How is mtDNA linked to evolution?
Research has been able to track the evolutionary changes in the mtDNA. We have different haplotypes of the mtDNA that has evolved over time, and looking at these haplotypes and changes, we're able to track back the original mitochondria and get an idea of how we've evolved out of Africa and dispersed throughout the world
366
True or false: Mitochondrial disease can be due to mutations in either nuclear or mitochondrial genomes
True
367
What types of diseases have been found to have defects of mitochondrial function in adults?
Diseases of aging (e.g. type 2 diabetes, Parkinson's disease, artheroscleoritc heart disease, stroke, Alzheimer's disease, and cancer)
368
Over 400 pathogenic mtDNA mutations have been identified to date. What types of mtDNA mutations have been identified and what do these mutations affect?
Point mutations and deletions, affect either oxidative phosphorylation or their expression
369
What can the severity of mtDNA diseases be modulated by?
Nuclear genes
370
What is mtDNA heteroplasmy and what does it cause?
Pathogenic threshold, where the ratio of mutated to WT mtDNA in a cell determines whether the the mutant shows a pathogenic phenotype
- Low heteroplasmy results in low number of mutations causing pathogenic phenotype
371
How does mtDNA heteroplasmy relate to mtDNA bottleneck?
Heteroplasmy can cause daughter cells to have lots of mutant mtDNA and not a lot of WT mtDNA, or vice versa.
So some mature oocytes have lower levels of mutant mtDNA and some have higher levels of mtDNA, so you can't always predict if a mother with mitochondrial mutations will pass those onto her children.
372
What is the familial trend with point mutations in mtDNA?
Maternally inherited, multiple family members affected
373
What is the inheritance trend with deletion mutations in mtDNA?
Rarely inherited
374
What is the phenotype trend with homoplasmic mutations?
Generally mild, affect single organ (all copies of mtDNA in a cell contain the same mutation)
375
What is the phenotype trend with heteroplasmic mutations?
Affect multiple organ systems
376
What is an NUMT? How many have been found?
Short-term nuclear mtDNA, there are 211 NUMTs detected in the human genome, 27 specific to humans. Can be pathogenic.
377
What is the link between NUMT % in nuclear genome and age?
As you age, number of NUMTs increases
378
True or false: NUMT genes are mutated more easily
- Explain why or why not
True; these genes are non functional so they're under less selective pressure
379
How can you get around determining whether mtDNA mutations are in the mitochondria or in NUMTs?
Design PCR primers for NUMTs (since the sequences are known)
380
What is mtDNA transfer/donation?
In mothers with defective mtDNA, they can transfer their nucleus to another oocyte with WT mtDNA (a form of IVF) which prevents transmission of serious mtDNA disease
381
Is CRISPR effective for targeting mtDNA?
No,CRISPR requires protein and RNA components but there's no efficient way to target RNA into the mitochondria
382
What are some alternative strategies to CRISPR for targeting mtDNA? (2)
1. Using target nucleases like TALEN, zinc finger and others to specifically eliminate mutant mtDNA
2. Doing mtDNA editing, such as base modifying with enzymes like cytidine deaminase
383
What are some challenges with mtDNA gene editing?
1. Limited by types of edits (low amounts of off-target are not deleterious though)
2. Off-target editing
3. Specific targeting of tissue of interest
384
Why is dosing critical for base editing in mtDNA?
High expression can lead to mt dysfunction
385
Somatic mtDNA mutations are present in what type of people?
Old people, accumulated with aging and has a higher frequency in age-related pathologies (e.g. Alzheimer's and Parkinson's disease, cancers)
386
Describe POLG mouse and interpretation of results
POLG mouse had mutated mtDNA which caused faster ageing/shorter lifespan meaning that mtDNA mutations result in increased apoptosis in ageing tissue and that ageing can occur independently of oxidative damage
387
What is the mt theory of ageing?
Mitochondrial dysfunction causes ageing and produces ROS which causes oxidative damage and mtDNA mutations, which causes mitochondrial dysfunction
388
What are replication disorders in mtDNA caused by? What does this result in?
Mutations in nuclear encoded proteins, causes not enough mitochondria being made or deletion disorders (mutations can lead to deletions of mtDNA genes)
389
True or false: Many different pathological phenotypes result from mutations in pol gamma genes
True
390
What inheritance pattern is found in Duchenne muscular dystrophy (DMD)?
X-linked recessive disease
