MT2 Review Flashcards
(322 cards)
1
Q
Genetic heterogeneity
A
A single phenotype is caused by mutations in multiple loci
2
Q
Pleiotropy
A
A mutation in a single gene leads to multiple phenotypes
3
Q
What are the three types of genetic heterogeneity?
A
- allelic heterogeneity
- locus heterogeneity
- modifier genes
4
Q
What is allelic heterogeneity?
A
The occurrence of more than one allele at a locus (different alleles in the same gene cause the same phenotype)
5
Q
What is locus heterogeneity?
A
The association of more than one locus with a specific clinical phenotype (mutations in different loci cause the same phenotype)
6
Q
What are modifier genes?
A
Genes that influence the phenotype of a primary gene mutation. Create additive effects
7
Q
Provide an example of modifier genes
A
Mutations in BRCA 1 and 2 increase the risk of developing breast cancer, while mutations in CHEK2 further accelerate this risk
8
Q
Provide an example of allelic heterogeneity
A
Cystic fibrosis. Over 1000 mutant alleles in CFTR gene causes CF
9
Q
What are the symptoms of CF caused by?
A
LOF mutation in CFTR that prevents proper function of the Cl- ion channel, which maintains the balance of salt and water on mucous membranes. This causes a mucosal buildup in the lungs
10
Q
What is a hypomorphic allele?
A
A partial LOF that results in milder phenotypes
11
Q
What is a null allele?
A
A complete LOF that results in a severe phenotype/complete set of phenotypes
12
Q
What may allelic heterogeneity cause?
A
- hypomorphic alleles
- null alleles
- alleles that confer a special property (SCA)
13
Q
What is an example of locus heterogeneity?
A
Retinitis pigmentosa caused by mutations in >100 genes that can independently cause it. Autosomal dominant, recessive or X-linked depending on the locus
14
Q
True/False? For Retinitis pigmentosa, multiple mutations are needed for the phenotype. Why or why not?
A
False. One mutation is needed for the phenotype because it is an example of locus heterogeneity, not modifier genes
15
Q
What is another term for pleiotropy?
A
Clinical/phenotypic heterogeneity
16
Q
True/False? CF is an example of allelic heterogeneity, but can also be affected by modifier genes
A
True. Examples are mutations in the mannose-binding lectin, glutathione-S-transferase, transforming growth factor-beta1, tumor necrosis factor-alpha, etc. genes.
17
Q
What is hyperphenylalaninemia?
A
An increased level of phenylalanine in blood due to its accumulation caused by a LOF mutation in phenylalanine hydroxylase or BH4, a cofactor
18
Q
True/False? PKU, if caught early enough, can be reversed
A
True. If caught before 4 weeks of age, the symptoms can be avoided all together with proper treatment
19
Q
What is enzymopathy?
A
A metabolic disorder resulting from deficiency or abnormality of a specific enzyme
20
Q
What are the symptoms of hyperphenylalaninemia?
A
Intellectual disability, seizures, and behavioural issues
21
Q
Describe the biochemical pathway affected in hyperphenylalaninemia
A
Phenylalanine, an essential amino acid, is converted into tyrosine by phenylalanine hydroxylase and BH4. Tyrosine is converted into L-dopa using BH4 as a cofactor, which is used to create dopamine
22
Q
What is an essential amino acid vs. a non-essential one?
A
Essential amino acids must be acquired through diet, whereas non-essential amino acids are synthesized in the body
23
Q
Where is BH4 used?
A
- conversion of phe to tyr
- conversion of tyr to L-dopa
- conversion of trp to 5-OH trp
24
Q
Compare the severity of the phenotypes of a deficiency of PHA vs. BH4
A
Deficiency in BH4 will have a more severe phenotype because it is used in more than one reaction
25
Classic and variant PKU are caused by a deficiency in (enzyme) and is treated with:
PAH; low phenylalanine diet (variant PKU has a less restrictive diet)
26
Non-PKU hyperphenylalaninemia is caused by a deficiency in (enzyme), and is treated with:
PHA; a less restrictive diet than with classic and variant PKU
27
Classic, variant, and non-PKU hyperphenylalaninemia are an example of what?
Allelic heterogeneity (they are all at the same locus and produce the same phenotype in varying degrees of severity)
28
Impaired BH4 recycling is caused by a deficiency in (enzymes). It is treated with:
PCD and DHPR; a low phenylalanine diet and L-dopa
29
Impaired BH4 synthesis is caused by a deficiency in (enzymes). It is treated with:
GTP-CH and 6-PTS; a low phenylalanine diet, L-dopa, and BH4
30
Impaired BH4 synthesis and recycling are an example of what?
Locus heterogeneity (they are at different loci but produce a similar phenotype)
31
What is the inheritance pattern of PKU?
Autosomal recessive
32
What is the prevalence of PKU?
1/2900
33
How is PKU tested for in newborn screening methods?
Blood is taken a few days after birth and the ratio of phe/tyr is analyzed
34
Why isn't PKU screening done right at birth?
The baby needs time to build up detectable [phe] levels because the mother's metabolism was working for the baby up until that point
35
Why are variant and non-PKU hyperphenylalaninemia less severe than classic PKU?
Residual activity of mutated PAH
36
PKU is more prevalent in which ethnicity? European or Asian
European (31%), Asian (25%)
37
What is a compound heterozygote?
Individuals have 2 different mutant alleles at the same locus
38
True/False? Most PKU patients are compound homozygotes
False. They are compound heterozygotes
39
What is the prevalence of impaired BH4 recycling or synthesis within the PKU population? What does this imply?
1-3%; their PAH is wildtype
40
Why do BH4 patients develop neurological problems on a low-phe diet?
BH4 is needed in other processes to make L-dopa and 5-OH-trp by tyrosine hydroxylase and tryptophan hydroxylase, respectively
41
Why is mass newborn screening for PKU justified?
- common (1/2900 live births)
- failure to treat has severe consequences
- treatment is effective if begun early in life
42
What is immunogenetics?
A branch of biology that studies the genetics of the immune system
43
How many immune cells does the average healthy adult have on average? How many are synthesized daily?
1.2-1.5 trillion cells; 1-2 billion cells
44
Why do immune systems have such a high turnover?
Maintain integrity of the immune system
45
What is an innate immune response?
General, nonspecific, first line of defense, rapid, creation of a cytotoxic environment or barriers
46
Which cells are involved in the innate immune response? What are their progenitor and stem cells?
Eosinophils, basophils, neutrophils, and monocytes, natural killer cells; myeloid progenitor cell and hematopoietic stem cell
47
Which cells are involved in the adaptive immune response? What are their progenitor and stem cells?
T-cells and B-cells; lymphoid progenitor cell and hematopoietic stem cell
48
What is the adaptive immune response?
An acquired, specific, slower-reacting but longer lasting response seen only in vertebrates
49
ROS are part of the __________ immune system
Innate
50
Where do T and B cells mature?
Thymus and bone marrow, respectively
51
How do B and T cells develop their diverse spectrum of cell-surface receptors?
VDJ recombination and junctional diversity
52
Humoral response
Mediated by B-cells with antibodies
53
Cell-mediated response
Mediated by T-cells
54
After maturation, where do B and T cells go?
They circulate throughout the body to secondary lymphoid organs (spleen, lymph nodes)
55
Describe the immune response mediated by B cells
An antigen-presenting cell digests a foreign protein and presents its antigens on its surface for recognition and binding by T-helper cells (MHC II). They secrete cytokines that stimulate B cells with immunoglobulins specific to the foreign peptide. B cells also independently recognize the antigens with their BCRs. Activated B cells proliferate and differentiate into either plasma cells (secrete antibodies) or memory B cells. Antibodies bind to the antigen and neutralize the pathogen
56
Describe the immune response mediated by T cells
An antigen-presenting cell digests a foreign protein and presents its antigens on its surface for recognition by T cells (MHC I). They differentiate into memory, cytotoxic, or regulatory T cells and lyse the APC
57
What is the difference between an immunoglobulin receptor and an antibody?
Same thing, except antibodies are free-floating
58
What enhances B cell receptor diversity and affinity for recognition of the foreign antigen?
Somatic hypermutation
59
What is the role of cytotoxic, regulatory, and memory T cells, respectively?
Destroy infected cells, modulate immune response, allows for quick recognition of re-encountered antigens
60
What are B cells named after? T cells?
Burma of Fabricius (lymph organ in birds); Thymus
61
What are the three origins of specificity in the B cell immune pathway?
1. Class II MHC
2. T-cell receptor
3. B-cell receptor
62
What regions make up the variable region on an antigen? Which chains are involed?
Variable (tip of light), joining (middle of light), and diversity (tip of heavy)
63
True/False? Only the heavy chain has a constant region
False. Both chains share a portion of the constant region
64
What does VDJ stand for?
Variable, diversity, joining
65
What is the Beyond One Gene-One Antibody Hypothesis?
There are multiple genes used to create every antibody
66
Describe VDJ recombination
In a singular progenitor B cell, one V, D, and J gene are picked at random from the germline DNA. The unpicked genes are completely deleted out of the B-cell DNA. These genes are transcribed and translated into the antigen. If low affinity, the cell undergoes somatic hypermutation and affinity maturation to make a higher-affinity antigen
67
VDJ recombination allows for how many different antibodies? Is this before or after cell exposure to an antigen?
100,000 to 1,000,000 antibody types alone before exposure to an antigen
68
What is junctional diversity?
Indels in VDJ genes allow for multiple B cells to have the same gene, just a different version of it. Ex. B-cell 1 has the V4 gene. B-cell 2 has the V4 gene, except it has undergone a deletion, effectively creating a new gene
69
Where are the B cells located when they have rearranged DNA?
Bone marrow
70
What is the difference between splicing and VDJ recombination?
Splicing is reversible because it only occurs in the mRNA. VDJ recombination occurs in the DNA itself, so it cannot be reversed once the cell is differentiated
71
Describe how somatic hypermutation works
Antigen exposure to the antibody induces somatic hypermutation and affinity maturation, where the V-segment genes undergo high mutation rates, leading to a wide range of binding affinities for the antigen. The most specific antibody is selected
72
Describe how multiple combinations of heavy and light chains can increase antibody diversity
The random assembly of heavy and light chains increases diversity because of the multiple potential combinations available
73
Compare the structure of the alpha and beta chains of T cell receptors (TCR)
Alpha: has a variable, joining, and constant region (outwards to inwards)
Beta: has a variable, diversity, joining, and constant region (outwards to inwards)
74
Compare the diversity of the alpha and beta chains in TCRs
- alpha has more V segments (70) than beta (50-70)
- alpha has more J segments (61) than beta (13)
- beta has more D segments (2) than alpha (0)
75
What mechanism contributes to TCR diversity?
VDJ recombination
76
What chromosome are the MHC genes located on? In what order do they occur?
Chromosome 6p; Class II, Class III, and Class I
77
How many genes make up the MHC segment? How long is it?
Over 200 genes; 4Mb
78
The significant variability in the ____ long Class I MHC region is due to _______
1.8Mb; extensive allelic diversity
79
What is the role of Class I MHC?
Forms complexes with foreign peptides, crucial for cytotoxic T cell recognition and response; essential for initiating cytotoxic T cell responses against infected or abnormal cells (kills the APC and other foreign cells)
80
How do some pathogens evade the immune system? What is it similar to?
Some viruses can escape detection by cytotoxic T cells by suppressing MHC Class I gene expression in host cells; cancerous immune evasion
81
What is the historical context of the MHC?
Skin grafts between mice were performed, and some took while others were rejected. This is largely due to differences in class I MHCs leading to transplant intolerance
82
What criterion must be met for a successful graft in the context of MHC?
Donor and recipient must have similar MHC region to be compatible
83
Describe the role of TAP1 and TAP2
They are MHC proteins on the cell surface that present peptides to T-cell receptors. They transport molecules that process foreign peptides and carry them to the endoplasmic reticulum
84
How are the heavy chains of an antibody connected?
Disulfide bond
85
How are the light chains connected to the heavy chains in antibodies
Disulfide bond
86
How many distinct antibodies do B cells have the potential to make? Where does this diversity come from?
100 billion; VDJ recombination, junction diversity, somatic hypermutation, and multiple combinations of heavy and light chains
87
Describe the role of RAG1 and RAG2
They are recombinases involved in VDJ somatic recombination
88
What is an immunodeficiency disease?
A disease in which the immune system does not function properly
89
How do IDs occur?
Occur due to the absence or malfunction of any components of the immune system (T cells, B cells, MHC, complement proteins)
90
What is a primary ID? What is its prevalence?
Genetic immunodeficiency due to a mutation; 1/10,000 and over 300 identified
91
What is a secondary ID? Provide an example
Somatic immunodeficiency due to external factors like radiation, infection, drugs; HIV/AIDS
92
Describe B-cell immunodeficiencies
- mainly affect B cells
- absence of B cells leads to susceptibility to recurrent bacterial infections
- lack of antibodies and Bm cells
93
Describe T cell immunodeficiencies
- affect T cells but also completely blocks B cell response
- may lead to SCID, which makes patient susceptible to opportunistic infections
94
Why are B cells affected by T cell IDs?
The humoral response is dependent on Th cells for B cell proliferation
95
How may RAG1/RAG2 SCID be treated?
Bone marrow transplants
96
What kind of mutation-phenotype relationship type does SCID express?
a) pleiotropy
b) allelic heterogeneity
c) locus heterogeneity
d) modifier genes
C. Multiple loci cause the same phenotype when mutated
97
The USIDNET is a research consortium established to advance research in what?
Primary immunodeficiency
98
Describe the genetic cause, presence/absence of T cells, B cells, NK cells, and the effect on TCRs and antibodies of X-linked SCID
IL2RG LOF mutation (cannot recognize IL2, which is a cytokine); absent; non-functional; absent; no T cell activation, defective B cell response
99
Describe the genetic cause, presence/absence of T cells, B cells, NK cells, and the effect on TCRs and antibodies of ADA-SCID
ADA (adenine deaminase) deficiency; absent; absent; absent; no TCR or antibody production
100
Describe the genetic cause, presence/absence of T cells, B cells, NK cells, and the effect on TCRs and antibodies of RAG1/RAG2 SCID
LOF RAG1/RAG2 mutation; absent; absent; present; TCR and BCR gene rearrangement fails
101
Describe the genetic cause, presence/absence of T cells, B cells, NK cells, and the effect on TCRs and antibodies of JAK3 SCID
LOF JAK3 mutation (cannot detect cytokines); absent; non-functional; absent; no functional TCRs, defective B cell response
102
Is cancer an immunodeficiency disease?
Yes, as most are able to avoid the immune system as it cannot detect them
103
Describe traditional cancer treatment therapies
Use of radiation or drugs targets proliferating cells, which may be cancerous or healthy, so side-effects are usually severe
104
Describe cancer immunotherapy
Uses the patient's own immune system to selectively detect and kill cancer cells, leading to less side effects
105
Describe the selection process for T cells. Where is this done?
First, the progenitor T cell must differentiate into many other cells with different receptors. They undergo positive selection for reactivity with MHC. Then, they undergo negative selection for reactivity for self-antigens. T cells with reactivity for foreign antigens are activated during infection, then they proliferate and acquire effector functions; thymus
106
How does KrasG12D relate to immunogenetics?
It is a neo-antigen formed due to an oncogenic hit which allows it to bypass the immune system
107
Describe the steps from normal tissue to established solid tumors due to escape from the immune system
1. normal cells undergo mutation to become cancerous
2. (early malignancy) cancerous cell presents a tumor-specific antigen (MHC Class I) that is then either :
2. a) recognized early by cytotoxic T cells and killed due to early dysfunction or anergy (phase 1)
2. b) recognized late by cytotoxic T cells and killed due to late dysfunction (phase 2)
108
What is PD-1 and how does it fit into the escape of cancer cells from the immune system?
PD-1 is a protein expressed by cytotoxic T cells that suppresses their function when bound to cancer cells, which allows them to go undetected by the immune system
109
What are the receptors involved in the late dysfunction (phase 2) of immune system cells? Are they found on immune cells or cancer cells?
CD38, CD39, CD101, TIM3; cancerous cells (they inhibit immune cells)
110
What is a population?
A local group of people sharing a common gene pool
111
What is a gene pool?
A set of genetic information carried by the members of a sexually reproducing population
112
What is population genetics?
The study of genetic variation and how genes and genotypes are maintained or change in populations, not individual matings
113
How can populations be described?
Age structure, geography, birth and death rates, allele frequencies
114
Compare the diversity of a population versus an individual
Greater diversity in a population
115
Compare the gene pools of two geographically distinct populations. Why is this relevant?
Likely also distinct; inferences drawn for one population cannot be applied to other populations
116
Why do we study population genetics?
- identification of genetic risk factors
- enhancement of preventative care
- promotion of health equity
- optimization of resource allocation
- advancement of personalized medicine
117
When do we use direct measurement of alleles vs. indirect?
When heterozygous phenotypes are different from homozygous dominant phenotypes, we use direct, as we're able to extrapolate their genotypes. Indirect measurement (HW equation) is used for complete dominance patterns
118
Which allele dominance pattern displays haploinsufficiency?
Incomplete dominance (blending)
119
How is codominance different from incomplete dominance?
Codominance is when both alleles are expressed equally, so both types of protein are being produced, while incomplete dominance depends on the amount of production of a single protein
120
Which dominance patterns are used for direct measurement?
Incomplete dominance, codominance, and X-linked recessive traits in males
121
Which dominance patterns are used for indirect measurement?
Complete dominance
122
Provide an example of a codominant gene
Glycophorin A -> M and N alleles can make MM, MN, and NN genotypes that directly relate to three phenotypes
123
What are the seven assumptions of HWE?
1. population is infinitely large (no sampling errors)
2. all genotypes are equally able to reproduce
3. random mating
4. no migration
5. no mutations
6. no mating between generations
7. all mating produce the same number of offspring who are equally fertile
124
How many allele types, phenotypes, and genotypes are possible for a complete dominance gene?
2, 2, 3
125
How many allele types, phenotypes, and genotypes are possible for an incomplete dominance gene?
2, 3, 3
126
How many allele types, phenotypes, and genotypes are possible for a codominance gene?
2, 3, 3
127
What are the HW equations for allele frequencies and genotype frequencies?
p + q = 1
p^2 + 2pq + q^2 = 1
128
What is genetic equilibrium? What does it explain?
Allele and genotype frequency remains constant from generation to generation; dominant alleles do not replace recessive alleles
129
What may increase observed phenotype frequency?
When a population is not in equilibrium, as genotype frequencies may change but allele frequencies stay constant
130
If F0 and F1 genotype frequencies are the same,
The population is in HWE
131
The frequency of carriers of deleterious allele is used to calculate what?
The risk of having an affected child
132
For X-linked traits, females carry ____ and males carry ________ of the alleles
2/3; 1/3
133
The number of males with an X-linked mutant phenotype equals _______________. What does this mean?
The allele frequency for the recessive trait; we can count recessive alleles in males
134
When can we not use direct measurement for X-linked traits?
When there is more than 2 alleles
135
How many genotypes are possible for ABO blood types?
6; AA, AO, AB, BB, BO, OO
136
Why is estimating the frequency of heterozygotes in a population important for genetic counseling?
Inform patients of potential risks in offspring
137
How much DNA do we share with other people? How many bases do we differ by?
99.99%; 3,300,000bp
138
What are the levels at which genetic variation may occur?
- chromosomal
- copy number (sub-chromosomal)
- single nucleotide level
139
True/False? Most genetic variations are neutral/do not cause disease/do not have clinical significance
True
140
Compare variant vs. mutation, polymorphism
Polymorphism implies benign and mutation is socially unacceptable (diseased people are mutants)
141
Common variant frequency
>1% allele frequency in a population
142
Rare variant frequency
<1% allele frequency in a population
143
What are the three possible effects of variants?
- pathogenic
- benign
- unknown
144
Describe small variation
- single nucleotide variant (SNV)
- small indel
- dynamic repeats
- microsatellites
- most common
- variable effects
- >3,000,000
145
Describe copy number variation (CNV)
Deletion or duplication of one or more exons
146
An SNV in the promoter usually causes:
No transcription of the gene
147
An SNV at the transcription start site usually causes:
No 5'cap attachment, so early mRNA degradation
148
An SNV in the 3'UTR usually causes:
A disruption in the polyA signal, so no polyA tail is added and thus, early mRNA degradation
149
Nucleotide 1 is (almost) always which base? Why? When is it not?
A; start codon is always AUG; when A is mutated
150
What is the proper nomenclature for a G on cDNA where it's located 3 bases after the start codon?
c.4G
151
What is the proper nomenclature for a C on cDNA where it's located 8 bases before the start of the second exon that starts at position 90?
c.90-8C
152
How are SNVs denoted using proper cDNA nomeclature?
c.(position number)A>G
153
How are intronic sequences numbered in cDNA?
Numbered according to the first or last coding nucleotide in the nearest exon and:
- the number of nucleotides downstream from the splice donor site (+)
- the number of nucleotides upstream from the splice acceptor site (-)
154
How are amino acids numbered for proper protein sequence nomenclature?
Numbered consecutively starting with the initiation codon (1)
155
What is the proper nomenclature for the substitution of a glutamine for a tyrosine at position 32?
p.Tyr32Gln
156
What effects may SNVs have on gene products?
Nucleotide substitutions:
- synonymous
- non-synonymous
- nonsense
- read-through
- splicing
Duplication/deletion:
- small
157
What is the correct nomenclature for a synonymous mutation at leucine 325?
p.Leu325=
158
What is the correct nomenclature for a missense mutation at leucine 112 into proline?
p.Leu112Pro
159
What is the correct nomenclature for a nonsense mutation at leucine 33?
p.Leu33Ter or p.Leu33*
160
What may occur to a protein with a nonsense mutation?
- premature stop (truncated)
- mRNA may undergo nonsense mediated decay
161
Describe nonsense mediated decay
Cells scan for truncated proteins by detecting residual mRNA binding proteins left on the transcript because of the ribosome's premature stop (it bumps them off as it goes). When they are detected, they are degraded, so the phenotype associated with nonsense mutations is usually LOF because the truncated proteins and regular proteins are absent
162
How are start codon variants disease-causing?
Disruptions on the start codon may cause the exon to shorten, as the start site is not detected by the ribosome
163
What is the correct nomenclature for a read-through mutation where the stop codon at 807 is substituted for a glycine and terminates 102 codons downstream?
p.Ter807GlyextTer102
164
What is a read-through mutation?
When the stop codon is mutated into an amino acid, causing an extended protein that terminates further downstream
165
Variants in the splice site donors and acceptors cause what?
Splicing variants such as extended exons, exon skipping, introns being included, or exons being partially spliced out
166
If the number of bases in an indel is not divisible by multiples of ___, it'll result in a ________________ mutation
3; frameshift
167
In a Sanger sequencing graph, if some bases overlap in their frequency, what does this mean?
Overlap means the individual is heterozygous for an indel that has caused a frameshift mutation
168
What is the correct nomenclature for a deletion of a single C 6 bases after the first nucleotide that changes glutamine to asparagine, causing a premature stop at a codon 8 positions downstream?
c.7delC, p.Gln3AsnfsTer8
169
What is the replication error rate per base?
10^-10/base/cell division (1 error/1.5 divisions)
170
How many bases a day per cell are damaged? How?
10,000-1,000,000; natural processes and mutagens
171
What are the possible modes of inheritance?
- autosomal recessive
- autosomal dominant
- X-linked recessive
- X-linked dominant
- pseudodominance (depends on other factors)
172
What are dynamic repeats?
A major cause of neurological disorders that is an expansion of a simple repeat in a coding or non-coding region. Often demonstrate reduced-penetrance alleles and variable expressivity
173
What is penetrance?
Not all people that genetically have the disease phenotypically have it
174
What is variable expressivity?
People who have the disease show different levels of severity
175
Dynamic repeats as a consequence of expansion can either result in _______ of function or ___________ function phenotypes
Loss; novel
176
An example of an expansion disorder is:
Fragile X syndrome, where wildtype individuals have 6-54 repeats, carriers have 55-200 repeats, and those affected have 200+
177
The risk of a premutation expansion is dependent on:
Sex of the individual and the size of the repeat
178
What is anticipation?
Some expansion repeats have an earlier age of onset every generation caused by increased expansions per gen. Usually only shown through one parent
179
Fragile X and Mytonic dystrophy show anticipation through the ____________ side
Maternal
180
Huntington's disease shows anticipation through the ____________ side
Paternal
181
What type of disease is mytonic dystrophy?
Expansion repeat
182
What kind of mutation is Thanatophoric Dysplasia?
A read-through mutation in FGFR3
183
What kind of mutation results in Nager syndrome?
A frameshift mutation caused by a deletion
184
What is an example of a somatic disease? Hereditary disease?
Proteus syndrome (not expressed everywhere); hereditary breast and ovarian cancer
185
What are microsatellites?
Repeat units of 2-55bp in length, also called short tandem repeats. Most do not cause disease
186
How are microsatellite alleles identified?
By the number of repeats they have
187
What are microsatellites useful for?
Forensics (DNA fingerprinting), relatedness (paternity, twin studies), and disease-gene genomic localization
188
How can microsatellites be used to identify aneuploidy?
If more than 2 markers (triploid) show up at a locus, it is more than likely that individual has aneuploidy for that chromosome. Likewise, if one marker shows up in a 2:1 ratio, then that shows there are 2 copies of the same repeat length and another copy of a different length
189
What is copy number variation?
A large deletion/duplication of an entire section of a chromosome mediated by repetitive sequences. It predisposes to non-allelic homologous recombination (NAHR)
190
What mutation is Charcot-Marie-Tooth (CMT1a) caused by?
A duplication of a gene caused by mis-alignment during recombination
191
What mutation is Hereditary Neuropathy with Liability to Pressure Palsies (HNPP) caused by?
A deletion of a gene caused by mis-alignment during recombination
192
Indirect repeats mediate _________________. This causes what disease?
Intrachromatid recombination (inversion); Haemophilia A
193
What is the most common cause of X-linked Duchenne muscular dystrophy? What is to blame for the de novo events?
Exon-level duplication/deletion of DMD gene; Alu elements
194
What are Alu elements?
They are transposable elements that cause non-homologous recombination in introns
195
Why are carriers of CF selected for?
Heterozygote advantage over Cholera, as bodily fluids are held on to
196
CF is an example of a:
LOF mutation
197
Achondroplasia is an example of a:
GOF mutation
198
Sickle cell anemia is an example of a:
Novel function mutation
199
Osteogenesis imperfecta is an example of a:
Dominant negative mutation
200
Hereditary persistence of fetal hemoglobin (HPFH) and lactose tolerance are an example of:
Mis-expression mutations
201
Holoprosencephaly is an example of:
Haploinsufficiency
202
CMT1a is an example of a:
Gene dosage mutation (duplication)
203
Describe the difference between recessive inheritance and haploinsufficiency
Haploinsufficiency is the inability of a carrier to exhibit normal function, producing a different phenotype than homozygous genotypes. Haploinsufficiency results from a dominant LOF allele
204
Residual activity correlates with:
Clinical severity
205
Normal severity correlates with:
No non-functional copy present
206
Carrier severity correlates with:
50% activity, but normal phenotype
207
Non-classical severity correlates with:
~30% activity, adult-onset symptoms
208
GOF variants are associated with (recessive/dominant) disorders
Dominant
209
In achondroplasia, homozygous dominance is _______________, so there is a bias towards _________________
Lethal; heterozygotes or homozygous recessive (normal)
210
Why are SCA carriers more common in Africa?
Heterozygote advantage over malaria
211
What is a dominant negative variant?
A variant that results in a protein that adversely affects the normal product encoded by the same gene within the same cell
212
What is osteogenesis imperfecta caused by?
A dominant negative variant in which abnormal collagen causes a more severe phenotype than if collagen wasn't present at all
213
What kind of mis-expression does HPFH exhibit?
Heterochronic
214
What is HPFH?
The persistence of fetal hemoglobin caused by deletions in the beta-globin locus
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Mis-expression variants are commonly (recessive/dominant)
Dominant
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Lactase expression exhibits what kind of mis-expression?
Heterochronic
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How may haploinsufficiency arise?
May be inherited or de novo
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Gene dosage variants are (recessive/dominant)
Dominant
219
What are Mendel's laws?
1. Law of segregation (anaphase I)
2. Law of independent assortment (prometaphase I)
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What is the exception to Mendel's second law?
Linked genes
221
What is genetic linkage?
The tendency of characters to co-segregate in a pedigree because they lie in close proximity on a chromosome
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What is a genetic marker?
A DNA sequence that has classifiable alleles that allow the marker to be tracked through families (microsatellites)
223
When 2 markers follow Mendel's second law, what proportions do the gametes form?
1:1:1:1 (marker 1 is found equally associated with both marker 2 alleles)
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When 2 markers are linked, what are the proportions of the gametes?
One allele from marker 1 is found associated with one allele from marker 2 over 50% of the time
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What is an uninformative marker?
When both parents have the same genotype or when one diseased parent is homozygous, so it is unknown which allele has been inherited from the diseased parent
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What is an informative marker?
When both the disease and the associated allele are inherited from one parent
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How are microsatellites used as markers for determining linkage?
PCR amplification, then gel electrophoresis to determine the size of each allele for each marker. Then, using pedigrees, their linkage is identified
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If both markers in the offspring have the same alleles as the parent, what do we call them?
Parental
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If two markers are linked, which chromosome type (parental/recombinant) is likely to show up more frequently?
Parental will be more frequent
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What is the recombination rate θ?
The probability of inheriting recombined alleles. Also known as the distance between markers in cM
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What is the probability of inheriting the parental alleles together?
1-θ
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A LOD greater than 3 indicates:
Linkage confirmed
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A LOD smaller than -2 indicates:
Linkage disproven
234
How can LOD score be increased to reach a score greater than 3?
LOD scores from linkage analysis of multiple families can be added together
235
What is a genetic map?
An ordered map of markers (mainly microsatellites) generated by genotyping large families
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For unlinked markers, disease will be ________________ with both marker alleles
Equally associated (found on a separate chromosome)
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Describe what will be observed if a disease allele is unlinked the marker alleles
You will see offspring with both marker alleles and a diseased phenotype in equal proportions (A+:C+)
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How may the location of a disease gene be located using marker alleles?
The use of multiple markers on the same chromosome allows for the ability to confirm the location of the disease gene (whether or not it is on the chromosome, **NOT** where on the chromosome it is, unless more markers are used)
239
Why can't we say for sure if a marker allele is linked to a disease?
A LOD score must be used to determine the statistical significance, even if parental offspring outnumber recombinants
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What are the uses of genetic linkage?
- used clinically before DNA seq. widely available to determine risk of being carrier, affected, etc
- positional cloning (identify genomic location of a disease gene without prior knowledge)
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If the θ between marker 1 and the disease gene is 7% and the θ between marker 2 and the disease gene is 5%, what is the total θ? What does this mean?
5 + 7 = 12%; 88% of gametes will be parental, and 12% recombinants
242
What part of the genome is focused on for clinical testing?
Exonic regions, as 85% of Mendelian variants are found there
243
What is Rhett Syndrome?
A de novo X-linked dominant syndrome found only in females due to their heterozygosity at the MECP2 gene. In males, it is lethal
244
Which kinds of mutations are not detectable with Sanger sequencing?
Deletions or duplications
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Of the two heterozygous variants found in Rett Syndrome patients, which is more likely to cause severe symptoms?
The nonsense mutation (c.799C>T, p.Arg267Ter)
246
What dominance pattern does Leigh disease show? How can we identify the genetic cause?
Autosomal recessive (parents are third cousins and are unaffected); homozygosity mapping
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What is homozygosity mapping?
A type of positional cloning that determines the likelihood of homozygous disease variant coming from the same individual (identical by descent)
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What is meant by identical by descent?
The alleles of a homozygous individual came from a mutation in one individual (ancestry) through consanguineous mating
249
Why is IBD mapping not always correct?
Both parents may be homozygous for the same genes (identity by state)
250
Which part of the chromosome is most commonly recombined?
Ends of the chromosome (amount of identity to the original chromosome diminishes with each succeeding generation)
251
What are the two techniques in IBD mapping?
- analysis of microsatellites (LOD score)
- genotyping
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Compare IBD vs. IBS (identical by state)
IBD: parents are heterozygous for the variant allele, but have common ancestry
IBS: parents are homozygous for the same markers, so all offspring will be, too
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Why is there no homozygosity in the middle of a chromosome?
Centromere
254
Regions of a chromosome that lack heterozygosity are:
IBD
255
Why do we see an increase in heterozygosity at the ends of chromosomes?
Recombination
256
How are genes prioritized when determining the location of the variant allele?
Because there are multiple regions that may be contributing to the phenotype, next gen sequencing must be utilized
257
What is the mode of inheritance for Kabuki Syndrome?
De novo autosomal dominant
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When NGS is used to sequence the genome of a Kabuki Syndrome individual, what may lead to the interpretation that they are heterozygous at one locus?
Presence of different SNVs in equal amounts (A=G, where G is the SNV)
259
What are the advantages and disadvantages of Dideoxy Sequencing?
Advantages:
- relatively quick
- position-dependent
- longer sequencing
- high accuracy
- no bioinformatics necessary
Disadvantages:
- low throughput
- labour intensive
- cost/base is expensive
260
What are the advantages and disadvantages of Next Gen Seq?
Advantages:
- high throughput
- cost/base is inexpensive
Disadvantages:
- slower
- position-independent
- short sequencing
- not as accurate
- extensive bioinformatic support required
261
Why is exome sequencing more efficient? What are its limitations?
Benefits:
- exons are only 2% of the genome
- fewer variants
- ~80% of Mendelian variants are estimated to be in exons
Limitations:
- not all variants are coding
- not all exons captured
- deletions/duplications are hard to detect
- inversions and dynamic repeat expansions not detected
262
How are cohorts used to determine mode of inheritance? What modes of inheritance may be identified?
The genotypes of multiple individuals with the same phenotype are compared to uncover overlap in their sequences; dominant or recessive
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How are trios used to determine mode of inheritance? What modes of inheritance may be identified?
Genotypes of parents and child are compared to determine de novo mutations; de novo dominant and identification of inherited recessive disorders
264
How may recessive disorders be identified?
If they are a result of a consanguineous mating, or if their genetic mapping shows that they are homozygous in that region (sometimes IBD)
265
How are autosomal dominant variants identified by mapping? Why?
Distantly-related people are used for mapping because they will share the smallest amount of common variants that aren't related to the disease
266
How are somatic variants identified?
Genetic map of unaffected vs. affected cells is compared to determine the variant
267
How do we reduce the number of variants under investigation when genome mapping?
- restrict to exome
- filter for rare variants (<1% frequency)
268
How many nonsynonymous, synonymous, and other (nonsense, fs, splice, non-stop) mutations does each person have on average?
135, 278, 65, respectively
269
What are the three considerations in variant classification?
- normal population prevalence (must be <1%)
- previous gene association with disease (mode of inheritance)
- SNV classification (predicted protein effect, computer-based prediction models)
270
If someone comes in looking for genetic testing for a specific disease, should we screen for other diseases while we're at it?
While 1-3% of the general population has genetic diseases, we should not look for them unless it is asked for by the individual
271
What are the advantages and disadvantages of whole genome sequencing (short read)?
Advantages:
- able to detect SNVs, CONs, dynamic repeat expansions, intronic variants, GC rich regions
Disadvantages:
- variant interpretation is challenging
- instrument capacity
- data storage
272
What are the advantages and disadvantages of long read genome sequencing?
Advantages:
- very long reads (15kbp-4Mbp)
- increased detection of structural variants, repeat expansions
- native DNA (allows for reading of methylation)
Disadvantages:
- expensive
- decreased throughput
- decreased accuracy
273
What functions do mitochondria serve?
- generate ATP for oxidative phosphorylation
- Apoptosis
- Cell signaling
- Metabolism
- ROS
- Immunity
- Ca2+ homeostasis
- Iron-sulfur biosynthesis
- Steroid production
274
Describe the evidence for the endosymbiosis theory of mitochondria
- Has its own unique DNA that is circular (mtDNA)
- Dedicated machinery for transcription and replication
- Ribosomes
- Lipids (cardiolipin found within mitochondrial membrane and other bacterial membranes)
275
Describe the structure of a mitochondria
- Inner membrane (bacterial membrane, forms cristae for more SA)
- IMS
- Outer membrane
- Matrix (DNA, ribosomes)
- Proteome is ~1500 proteins
- Lipids (cardiolipin in inner membrane)
- mtDNA only encodes 13 proteins, the rest are imported from cytosol
276
How is mtDNA organized in a human mitochondria?
- Compact
- 100's-1000's copies per cell
- Nucleoid (packaged by mtTFA, analogous to histones)
- Expression and replication is regulated by nuclear-encoded proteins (nucleus of **cell**)
277
Where do the proteins of the ETC originate?
Some from mtDNA, some from nuclear DNA
278
What is a mitochondrial network?
A network of mitochondria throughout the cell connected by proteins
279
List the differences between the nuclear and mitochondrial genome
Nuclear:
- linear
- 3.3Gb
- 20k genes
- introns and other non-coding sequences
- monocistronic
- different codons
Mitochondrial:
- circular
- 16.5kb
- 37 genes
- minimal non-coding and no introns
- different codons
- polycistronic
- maternal inheritance
- 10x mutation rate and lower repair rate
280
Why is the mutation rate for mtDNA so high?
The mitochondria produces a lot of ROS, which cause damage to the DNA
281
What does the mitochondria use for its start codon?
AUA and AUG both code for Met
282
What does the mitochondria use the codon UGA for?
Instead of a stop codon, it codes for Trp
283
What does the mitochondria use as stop codons?
AGA and AGG (usually code for Arg)
284
Why can't mitochondrial DNA be inserted and used in the nucleus?
Because it uses different codons than us, so the proteins will be different
285
What is TFAM?
A DNA-binding protein that is used for compaction of mtDNA. It bends it into a U-shape to package it
286
What are the mtDNA replicative machinery proteins?
- POLγA
- POLγB
- TWINKLE (analogous to helicase)
- mtSSB (stabilize ssDNA)
287
What is the D-loop?
A displacement loop caused by the displacement of the heavy strand (GC rich) by a 7S strand for transcription initiation. Forms a triple helix
288
Describe the steps for mtDNA transcription
1. Heavy strand displaced (GC rich)
2. HSP1 starts early, then terminates early
3. HSP2 transcribes whole genome
4. LSP transcribes whole genome in other direction (transcription on both strands)
289
How is the transcription of mitochondrial rRNA made more efficient?
The genes for 12S and 16S rRNA are bent into a loop so that the transcriptional machinery may go around the loop as many times as needed
290
What is the tRNA punctuation model?
In mtDNA, the genes for tRNA are interspersed within the mRNA ORFs. Because mtDNA is polycistronic, all of these genes are transcribed at once. In order for tRNA to mature, it must be cut by an endoribonuclease on its 5' and 3' ends, freeing the adjacent mRNA at the same time
291
Is epigenetics involved with mtDNA?
It's complicated. Methylation has been found on CpG, CpA, CpC, and CpT islands, but it is likely that this methylation is an artefact of methods used to estimate methylation. Mitochondria also lack a methyltransferase.
TFAM may be phosphorylated or acetylated, but we do not know the functional consequence yet
292
Describe genome inheritance within the mitochondria (mitochondrial inheritance)
The mitochondrial network is fused together, which allows for the proper distribution of mtDNA throughout the network. Each mitochondria has one copy of the genome and they divide by fission, initiated by the ER
293
What does the mitochondrial network look like in G1-S phase?
The mitochondria are fused so they appear elongated
294
What does the mitochondrial network look like in G2-M phase?
The mitochondria have divided by fission, so they are fragmented
295
What may happen if the mitochondria are still fused by the time its host cell divides?
The mtDNA may be unevenly distributed between the two daughter cells
296
Describe mitochondrial inheritance on the organismal level
Maternally inherited. Haplotypes may be used to track the spread of humans when they evolved
297
How are mitochondrial diseases characterized?
Dysfunctional mitochondria
298
True/False? Mitochondrial diseases may occur only if the mtDNA is mutated
False. Both nuclear and mtDNA may cause dysfunction
299
How prevalent are mitochondrial diseases?
1/4000 by the age of 10 years
300
Mitochondrial dysfunction is known to have implications in _____________. This also includes diseases such as (provide 6 examples)
Aging;
- type II diabetes
- Parkinson's
- atherosclerotic heart disease
- stroke
- Alzheimer's
- cancer
301
What common drugs may affect mitochondria? Why?
Antibiotics; they are from bacterial descent
302
What is the prevalence of inherited mtDNA mutations?
1/4300
303
What mutations may cause a pathogenic phenotype in mtDNA? Why?
Point mutations and deletion; affect oxidative phosphorylation or other protein expression
304
How are the phenotypes of mtDNA mutations modulated?
Severity depends on the nuclear genes
305
What is heteroplasmy?
An accumulation of mtDNA mutations before pathogenic affect (some, but not all, mitochondria are dysfunctional within a cell)
306
What is the pathogenic threshold?
The ratio of mutated/wildtype mitochondria in a cell before it becomes pathogenic
307
Describe the mtDNA bottleneck
During cell division, dysfunctional mitochondria are randomly distributed. Some mature germline cells may receive more dys. mt than others, so the pathogenic threshold is met sooner
308
Homoplasmic mutations
Generally mild and affect a single organ
309
Heteroplasmic mutations
Severe and affect multiple organ systems
310
What is the inheritance pattern in point mutations within mtDNA? What evidence supports this?
Maternally inherited; multiple family members are affected if they share common ancestry through the mother
311
What are NUMTs?
mtDNA that has been integrated into the nuclear genome
312
How many NUMTs do humans have? How many are specific to just humans?
211; 27
313
Describe mtDNA transfer
A three-parent embryo is created by taking the nucleus of the mother's egg and implanting it in an enucleated donor egg. This gets fertilized like normal
314
What is mtDNA transfer used for?
If the mother has a mitochondrial disorder, it is possible to avoid passing on those genes to her young through this method
315
Explain the controversy behind three-parent embryos
People believe that because there is DNA from 3 individuals, it is unethical or not morally right, but in reality, only 0.054% of the offspring's DNA is composed of the donor's DNA
316
What are the alternative strategies to CRISPR for mtDNA editing? How do they work?
TALEN or zinc fingers recognize a sequence in the mtDNA and cleave it (like CRISPR), which linearizes it, causing the DNA to degrade. This may be used to change the ratio of dysfunctional/wildtype mitochondria in the cell, below the pathogenic threshold
317
What is base editing in mtDNA?
A base is modified so it base-pairs with a different molecule than usual. This is not fixed, so once the mitochondria undergoes replication, the mutant mtDNA is passed on with the new base pair
318
What limits base editing in mtDNA?
- still has off-target effects (targeting is tricky)
- high expression may lead to mitochondrial dysfunction
- dosing is critical
319
What is observed in specimens where their POLγ is mutated so its exonuclease activity no longer works?
Homozygotes for this gene showed rapid aging effects due to more damage to their mtDNA
320
What is the mitochondrial theory of aging?
1. Mitochondria produces ROS
2. ROS damage mtDNA, causing mutation
3. Eventual mitochondrial dysfunction
4. Repeat 1-3
5. Aging
321
What are mtDNA maintenance disorders?
Nuclear-encoded proteins responsible for mtDNA replication may cause deletions if they are impaired
322
What is an example of a mtDNA maintenance disorder?
Impaired function of POLγ, as it is encoded by nuclear DNA
