Midterm 1 Flashcards
(268 cards)
1
Q
According to WHO, it is now universally acknowledged that DNA determines not only the congenital malformations that kill millions of children, but also…
Provide an example
A
Predisposes people postnatally to mental illness and major non-communicable disease such as cancer, cardiovascular disease, hypertension, asthma, diabetes, rheumatoid arthritis, etc.
Ex: In individuals with a single copy mutation in a gene called adenomatous polyposis coli or APC (a hereditary condition named familial adenomatous polyposis or FAP), there is a nearly 100% chance of developing colorectal cancer by 39 years old if the condition is left untreated.
2
Q
What are the five roles of modern medical genetics?
A
- Identify and characterize mutations that cause genetic diseases
- Understand how mutations affect health
- Improve diagnosis
- Improve disease management
- Find a cure
3
Q
The degree of impact of genetics on individual diseases varies from what 3 levels?
A
- Entirely causative
- Major role
- Minor role
4
Q
Describe how genetics could play an entirely causative role in individual diseases
A
You have the mutation, you will get the disease
- e.g. single gene disorders (like cystic fibrosis), chromosomal disorders (like down syndrome)
5
Q
Describe how genetics could play a major role on individual diseases
A
Major risk conferring genes, several mutations that make us vulnerable to certain diseases.
- e.g. carriers for BRCA1 mutations have a 30% chance of developing ovarian cancer
6
Q
What genetic disorders play a minor role on the impact of genetic diseases?
A
Multifactorial models, severity of the disease is impacted by many factors
- e.g. spina bifida (neural tube defect that affects the spine)
- lots of different genes could potentially be involved in the development of the disease.
7
Q
What is the most common type of genetic disease? Which is the least common?
A
Most common: multifactorial
Least common: chromosomal
8
Q
__% of children by age 5 will have genetic disorders, while __% of people will have acquired a genetic disease throughout their entire life
A
5, 67
9
Q
Continuum of disease causation
A
Environmental (e.g. influenza, measles, infectious diseases) to genetic (cystic fibrosis, hemophilia A)
- diabetes and heart disease fall in between
10
Q
True or false: identifiable multifactorial genetic causes of childhood death have been on the rise
A
True
- partly due to increased frequency of genetic analysis
11
Q
Multifactorial disorders are believed to account for approximately 1/2 of…
A
All congenital malformations
12
Q
True or false: multifactorial disorders also include common chronic disorders of adulthood with genetic components like hypertension, rheumatoid arthritis, etc.
A
True
13
Q
True or false: multifactorial diseases do not include any mental disorders
A
False; multifactorial diseases include many common psychological disorders of childhood, including dyslexia, ADHD and specific language impairments.
14
Q
What are 5 components of modern medical genetics?
A
- Inheritance of diseases
- Disease gene identification
- How mutations affect health (molecular mechanisms)
- Diagnosis and treatment of genetic disease (DNA based diagnosis for several thousand inherited conditions; gene therapy for some)
- Genetic counselling
15
Q
What are 6 components of clinical genetics?
A
- Diagnosis (prenatal, pediatric, adults)
- Prognosis (likely course and clinical outcome of a disease)
- Management
- Risks assessment/counselling
- Reproductive options (in some cases, genetic mutations limit reproductive options)
- Support
16
Q
What is likely to be the greatest growth area for genomic medicine?
A
Diagnosis in adults
17
Q
What is the promise regarding genomic medicine?
A
To identify individuals with genetic predispositions to:
- cancer, heart disease, stroke, diabetes, etc
- be able to do primary prevention via targeted risk reduction (losing weight), early use of medical therapy (treat borderline hypertension), and gene therapy.
18
Q
What are three potential treatment methods when needing to target a gene? What is important to note about the viral options?
A
- DNA viruses
- RNA viruses
- Liposomes
Viral options can be carcinogenic or induce an immune response
19
Q
True or false: the cost for sequencing the human genome is decreasing
A
True
20
Q
What is the definition of a gene?
A
A segment of DNA that contains the necessary code to instruct the production of proteins or molecules that perform specific function(s) in the body.
21
Q
Direction of transcription RELATIVE TO THE CODING STRAND/UPPER STRAND is…
A
5’ to 3’
22
Q
Housekeeping genes
A
Gene products are needed in almost every cell, and are constitutively expressed.
- e.g. genes encoding ribosomal proteins, RNA polymerases, tRNA synthesizing genes, heat shock proteins, histones, etc.
23
Q
Tissue/cell type-specific genes
A
Highest expression in certain cells (opposite of housekeeping genes)
24
Q
Enhancer
A
Often several hundreds or thousands of base pairs away from promoter, also influence transcription through activators and co-activators
25
Activators vs co-activators
Activators bind directly to enhancers, co-activators don't have a DNA-binding domain and bind to activators, other coactivators and transcription factors
26
Silencer
Often several hundreds or thousands of base pairs away from promoter, influences transcription by binding to proteins that block transcription
27
RNA pol II
Carries out transcription, through interaction with a large complex of about 50 other proteins assembled at the core promoter
28
RNA pol II binding to the promoter is (specific/non-specific)
- Explain the significance of this
Non-specific (it can bind to any gene)
- Why other proteins at the promoter are required to recruit RNA pol effectively
29
Basal/general transcription factors
Required for RNA polymerase to function at a transcription site, such as TFIIA and TFIIB
30
Specific transcription factors
Modulate transcription in a specific context, for example certain developmental stage or differential regulation
31
The promoter sequence is located on which strand?
Coding
32
What are the three main post-transcriptional modifications?
1. Intron splicing
2. 5' cap
3. Poly A tail
33
5' cap on mRNA allows for...
Protection of the mRNA until translation
34
5' cap is specifically...
7-methylguanosine (m7G)
35
The m7G cap forms a...
5'-5' phosphate bridge
36
The m7G blocks the possibility of any...
Addition to the 5' end of the mRNA
37
What are the 2 genomes?
1. Nuclear DNA
2. mtDNA
38
mtDNA is (linear/circular)
Circular
39
True or false: mtDNA contains introns
False, mtDNA lacks introns
40
mtDNA is exclusively (maternally/paternally) inherited
Maternally
41
How many genes are in mtDNA?
37 (2 rRNAs, 22 tRNAs and 13 proteins for mitochondrial oxidative phosphorylation in cellular respiration)
42
Which of the following about enhancers is true?
a) These are DNA sequences that regulate the transcription of certain genes
b) These are non-coding DNA sequences present in between two exons
c) these are proteins that control gene function
d) they associate with activators and co-activators during gene transcription
e) A and D
E
43
mtDNA consists of ___ and ___ strands
Heavy (H) which is the outer strand and light (L) which is the inner strand
44
D (displacement loop) in mtDNA?
A triple-stranded region due to a short third strand called 7S DNA
- Acts as a promoter
- Contains the origin of replication for mtDNA
- 7S DNA can base-pair with the L-strand
45
True or false: mtDNA replication occurs with nuclear DNA replication
False; mtDNA replication is variable between cells (i.e. distinct from the nuclear DNA replication)
46
True or false: mtDNA contains 2 copies of each gene in diploids
False. There are between 1000 and 10,000 mtDNA copies within the inner mitochondrial compartment (matrix) in a cell (except Oocytes, which contain 100,000 mtDNA copies)
47
Maternally inherited diabetes and deafness (MIDD)
Disease due to defects in mtDNA
- 1% of diabetic patients and most commin in the Japanese population
- mutations in mitochondrial genes. MT-TL1, MT-TK, or MT-TE by REDUCING TRNA ACTIVITIES
48
Human Genome Project (HGP)
An international, collaborative research program aimed to map and understand all the genes (genome) of human beings
- we already knew the general structure of the genome but we didn't know how many actual genes were in the genome
49
Steps for sequencing the human genome (4)
1. Isolate genomic DNA from thousands of cells
2. Fragmentation of DNA (physical dissociation, enzymatic dissociation or sonication)
3. Capture the pieces to sequence them using bacterial plasmids, which are cloned into bacteria/BACs (1 clone/bacteria, work as "sponges" to take all the DNA fragments in)
4. Using computer algorithms, order of sequenced DNA fragments is determined (by identifying overlapping regions between fragments)
50
What were the most surprising findings from the HGP?
- 3.1 Gb of DNA (haploid)/individual
- <1.5% encodes proteins
- ~45% is unique/single-copy (non-repetitive) DNA
- the rest is repetitive DNA that is difficult to sequence (still trying to determine the role of repetitive DNA)
51
True or false: exon frequency is low compared to that of introns
True
52
Intergenic single copy DNA
Located between the genes (e.g. contains the enhancers)
53
When sequencing the human genome, different individuals will have different SNPs. How is the consensus/reference genome determined?
Consensus sequence/reference genome is determined by statistically determining the most common nucleotide at each position
54
Pseudogenes
Sequence is similar to already identified genes but we don't know much about these
55
True or false: Human genome annotations (i.e. finding reference sequences) are fixed
False; they are variable.
- Based on probabilities of certain nucleotides being at given positions based on sequenced samples
56
What 5 things are required for a Sanger sequencing reaction?
Template DNA, primer, DNA polymerase, dNTPs, ddNTPs
57
What is something that can be done with Sanger sequencing to not require 4 separate reactions (wells)?
Can label each ddNTP with 4 different colours so don't need 4 different reactions and can put all the ddNTPs into the same reaction
- Scanner records coloured images of different sized termination fragments for each fluorescent-labelled ddNTP
- Computer processes fluorescent signals to generate an electropherogram, assigning a base to each peak
58
Phred (Phil's revised editing program)
Predicts errors in sequencing since electropherogram data is messy
59
Automated sequencers
Able to do thousands of reactions at the same time.
60
Why are there a different number of annotated genes between different databases?
Due to conjoined genes
61
Conjoined genes
Due to read-through transcription, sometimes two adjacent genes form either two mRNA molecules or one complete mRNA molecule; some people interpret this as 1 gene, while others interpret it as two
- How many transcripts should be counted? This is a complicated issue
62
By 2013, 92% of the human genome was sequenced. Why was it so hard to sequence the entire genome?
Due to the presence of heterochromatin
63
True or false: During the human genome project, researchers could only read 500 bases at a time. Now they can read up to 100,000
- explain why or why not
new sequencing methods invented in 2020, so eventually 100% of the human genome was sequenced.
64
What are the 3 types of DNA?
1. Single copy DNA
2. Dispersed repetitive DNA
3. Satellite DNA
65
What is single-copy DNA? What percentage of the genome does it make up?
Unique regions in the genome that don't exist anywhere else in the same genome
- 45%
66
What is dispersed repetitive DNA? What percentage of the genome does it make up?
Relatively larger fragments repeated throughout genome
- 45%
67
What is satellite DNA? What percentage of the genome does it make up?
Relatively smaller fragments repeated throughout the genome
- 10%
68
What are the three families of satellite DNA?
1. Alpha-satellite
2. Minisatellites
3. Microsatellites
69
Alpha-satellite
171-bp sequence that can extend (repeat back to back) to several million base pairs or longer
- Near the centromeres of chromosomes
70
Minisatellites
- Blocks of tandem repeats (each 11 bp long or greater)
- Total length of a few thousand base pairs
71
Microsatellites
- The repeat units are 1 to 10 bp long
- Total length less than a few hundred base pairs
- Generally present in clusters
72
Why are minisatellites and microsatellites of special interest in human genetics?
They vary in length among individuals, making them highly useful for forensic identification and gene mapping
73
How can microsatellites be applied to cancer diagnosis?
Microsatellite sequencing in cancers can predict clinical outcome by comparing microsatellites between cancerous and normal tissue. If different, microsatellites are unstable.
74
What are the three families of dispersed repetitive DNA?
1. SINE (Short Interspersed Nuclear Elements)
2. LINE (Long Interspersed Nuclear Element or L1)
3. Segmental duplication family
75
SINE
- 1% of genome (>1 million members)
- Each member is ~300 bp in length
- e.g. Alu family
76
LINE
- 20% of the genome
- Members could be as long as 6 kb in length
77
Segmental duplication family
>5% of the genome
- Can span hundreds of kb
- include DNA blocks that typically share >90% identity
78
True or false: some Alu sequences and LINEs can generate copies of themselves, which can then be inserted into other parts of the genome.
True
- This is how they have achieved such remarkable numbers.
- These insertions can sometimes interrupt a protein-coding gene, causing genetic disease
79
Which of the following statements is true regarding the introns?
a) Introns are always located between the TATA box and the promoter region of a gene
b) introns are not spliced out from protein-coding mRNA Molecules before translation
c) Presence of introns distinguishes eukaryotes from prokaryotes
d) Intronic region of mRNA is stabilized by a modified m7G cap before translation
e) A and C
C
80
DNA is wound around a histone protein core (__ histones) to form a ___
8, nucleosome
81
The nucleosomes form a ____ _____; each turn of the ____ includes ___ nucleosomes
The nucleosomes form a helical solenoid; each turn of the solenoid includes six nucleosomes
82
The solenoids themselves are organized into _____ ______
Chromatin loops
83
What is the highest level of DNA packaging?
Chromosomes
84
Chromosomes can only be visualized during...
Cell division
85
Order the parts of the mitosis (somatic) cell cycle in order of longest to shortest
G1, S, G2, M
86
What happens to the cell in G2?
The cell stops making new DNA and makes proteins needed for cell division
87
What phase of the cell cycle is the cell in 50% of the time?
G1
88
In what phase of mitosis is the greatest chromosome condensation seen?
Metaphase
89
In what phase of the cell cycle do spindle fibers form?
Prometaphase
90
The short arm of a chromosome is also called the __ arm
p
91
The long arm of a chromosome is also called the __ arm
q
92
How are human karyotypes visualized?
Using Giemsa banding (G-banding)
93
After using G-banding to visualize chromosomes, the light regions refer to the (euchromatin/heterochromatin) while the dark regions refer to the (euchromatin/heterochromatin)
euchromatin, heterochromatin
94
True or false: each chromosome has a very unique banding pattern
True
95
What are the three chromosome classifications?
1. Metacentric
2. Submetacentric
3. Acrocentric
96
Metacentric chromosome
Centromere is located near the middle (not exactly the middle, so there are still p and q arms)
97
Submetacentric chromosome
Centromere is between middle and tip-telomere)
98
Acrocentric chromosome
Centromeres are near the tip
99
What are the two unique features of acrocentric chromosomes?
1. Stalk
2. Satellite
100
Stalk region of acrocentric chromosome
Contains ribosomal genes, and forms the nucleolus in interphase
101
Satellite region of acrosome
A non-coding region (provides protection like telomeres)
102
True or false: acrocentric chromosomes have telomeres
False
- Satellites replace telomeres on acrocentric chromosomes
103
True or false: the size of chromosomes correlate directly with the number of protein-coding genes
False
- They roughly correlate, but not strictly
104
How is the number of chromatids within a cell often indicated?
By the letter c
105
One single chromosome (haploid) that is unreplicated would be written as...
1n 1c
106
Two homolog (diploid) unreplicated chromosomes would be written as...
2n 2c
107
One single chromosome (haploid) in which DNA has been duplicated would be written as...
1n 2c
108
Two homolog chromosomes (diploid) consisting each of two sister chromatids would be written as...
2n 4c
109
A cell with two homologs before meiosis I would be written as...
2n 4c
110
Cells that originally contained two homologs before meiosis I would be written as what after meiosis I?
1n 2c
111
A cell with one duplicated haploid after meiosis I would be written as what after meiosis II?
1n 1c
112
A cell with two chromosomes would be written as what before and after mitosis
Before mitosis: 2n 4c
After mitosis: 2n 2c
113
Meiosis produces __ cells from __, while mitosis produces __ cells from __
Meiosis: 4, 1
Mitosis: 2, 1
114
What are the 4 genetic consequences of meiosis?
1. Shuffling of the genetic materials by recombination
2. Additional shuffling of the genetic material by random assortment of the homologues (different chromosome chosen from each pair of homologues in eggs/sperm)
3. Reduction of the chromosome number from diploid (2n) to haploid (n)
4. Segregation of alleles (alternative forms of a single gene)
115
Genes that encode major components of the molecular machinery that synthesizes proteins from mature mRNA molecules
a) are contained in Stalk of acrocentric chromosomes
b) are contained in Satellite of acrocentric chromosomes
c) are located on X chromosome to ensure expression in males and females
d) are specifically present on p arm of the submetacentric chromosomes
e) none of the above
A
116
Meiotic nondisjunction
When the homologous chromosomes OR THE SISTER CHROMATIDS of a chromosome failed to separate from one another to travel to the opposite poles, resulting in cells that do not have a normal number of chromosomes
117
Give an example of a good mutation
Humans were lactose intolerant, but in some populations a mutation occurred (SNP) that induced lactose tolerance
- Surprisingly far from lactase gene but still had effect, on chromosome 2
- C nucleotide= lactose intolerance, T SNP resulted in lactose tolerance
118
Non-disjunction can occur at either the first or second division of meiosis. XYY individuals would most likely arise form nondisjunction at the ___ meiotic division in the ____
- Draw the effects
Second, father
119
Non-disjunction can occur at either the first or second division of meiosis. XXY individuals would most likely arise form nondisjunction at the ___ meiotic division in the ____
- Draw the effects
First, father OR second, mother OR first, mother
120
What is cytogenetics?
The study of structure and properties of chromosomes
121
Define clinical cytogenetics
The practice of medical genetics by studying the structure and number of chromosomes to identify chromosome abnormalities (disorders)
122
Aneuploidy
One or more extra or missing chromosomes
123
Polyploidy
Possessing more than two complete sets of chromosomes
124
What is the leading cause of pregnancy loss?
Chromosomal abnormailites
125
What % of oocytes have an extra or missing chromosome?
20-25%
126
Trisomy 21
Down's syndrome
127
Trisomy 18
Edward's syndrome
128
Trisomy 13
Patau syndrome
129
What % of conceptions will have chromosomal abnormalities? What % of them are lost before term?
10-20%
- 95% of them are lost before term
130
Why are other trisomies (e.g. trisomy 1, 2, etc) not found?
These trisomies are lethal
131
What are the two types of balanced chromosomal rearrangements?
1. Robertsonian translocations
2. Reciprocal translocations
132
What % of pregnancies is women > 35 years have chromosomal abnormalities
2
133
What are 4 clinical indications for chromosomal disorders and give examples
1. Problem with early growth and development
- developmental delay
- physical malformations
- ambiguous genitalia
- mental retardation
2. Stillbirth and neonatal death
- 10% of stillbirth (survived 28 weeks) is due to chromosome abnormalities
- 10% of neonatal (from birth to 4 weeks) deaths are due to chromosome abnormalities
3. Fertility problems
- amenorrhea (i.e. abnormal menstruation)
- Recurrent miscarriage
- History of infertility
4. Family history:
- a known or suspected chromosome abnormality in a first-degree relative
134
What are the two aims of karyotyping?
1. To determine abnormal chromosomal numbers caused by defective duplication and/or segregation
2. To determine abnormal chromosomal structures caused by rearrangements
135
What two types of abnormal chromosome structures can be detected through karyotyping? Provide examples for each
1. Insertion/deletions (InDel)
- Autosomal deletions occurs at 1 in 7,000 live births
- e.g. cri du chat syndrome (partial deletion of 5p)
2. Translocation (the transfer of a segment of one chromosome to another chromosome)
- e.g. Robertsonian translocation
136
Describe Robertsonian translocation
A translocation between 2 acrocentric chromosomes (i.e. centromere near one end of the chromosome) by fusion at or near the centromere, with the loss of the short arms
137
True or false: Robertsonian translocation is usually linked to a disease
- Explain why or why not
False; you will still have the stalks of other chromosomes, so ribosomal machinery will still be made
138
Terminal deletion
A deletion that occurs towards the end of a chromosome
139
Interstitial deletion
Part of chromosome deleted within (needs 2 breakage sites)
140
Chromosomal insertion
Gene fragment goes from one chromosome to another
141
How are translocations different from chromosomal insertions
Fragment added towards the end of a chromosome in translocation
142
What are the two requirements for cells in routine cytogenic (karyotyping) analysis? Based on these requirements, what cell is typically used?
Requirements:
- Cells must be easy to obtain
- Cells are able to grow rapidly in culture (because chromosomes condense in mitosis so need cells to be dividing)
Because of this: white blood cells (T lymphocytes) used
143
What are the 6 steps for routine cytogenetic (karyotyping) analysis
1. Venipuncture and then add heparin (anti-coagulant since blood has a tendency to clot easily)
2. Isolate white blood cells (T LYMPHOCYTES), and culture them
3. Add colcemid, which arrests cells in metaphase by degrading spindles after chromosomes align at metaphase plate
4. Place cells in hypotonic solution to swell them. This allows the chromosomes to burst open gently, allowing for the spreading of chromosomes onto a slide
5. Fixation (neutralize protein activities such as apoptosis and kinase activities) + staining to prepare for microscope imaging
6. Squish the cells and analyze them
144
Giemsa banding (G-banding) is the most commonly used method for cytogenetic analysis. It allows for (light/dark) staining at the GC rich regions, and (light/dark) staining at the AT rich regions
Light, dark
145
Describe the composition of Giemsa stain
Nucleic acid stain; mixture of methylene blue, eosin, and Azure B
146
What is an ideograph?
A visual representation of chromosomes that shows their size, banding patterns, and cytogenetic landmarks
147
Describe Q (Quinacrine) banding, and what it is used to detect
- Staining with quinacrine mustard
- Used to detect heteromorphism, a morphological variant of a chromosome (to detect homologous chromosome pair which are not morphologically identical)
148
Describe R (Reverse) banding and what it is used to detect
- Is the standard method used in Europe
- Specimens are heated before staining
- The resulting dark and light bands are the reverse of G and Q banding methods (GC=dark like in telomeres and AT=light)
- Used to analyze the distal ends of chromosomes that stain poorly with G or Q banding methods
149
Describe C banding and what it is used to detect
- Is the specific method involving Xylene substitute for staining
- Detects centromeric regions and regions constaining constitutive heterochromatin, a type of condensed chromatin lies near the centromere, and staining darkly in interphase cells
150
Describe high-resolution (prometaphase) banding and what it is used to detect
- Use G or R banding method to stain chromosomes that are at early mitotic stage (i.e. before they reach maximum condensation and are more extended)
- Chromosomes that look longer when arrested result in more bands shown, so this is especially useful for identifying subtle structural abnormalities of chromosomes (higher resolution)
151
Cells are generally arrested at which stage of the cell cycle for use in karyotyping?
Metaphase
152
Describe Fluorescence In Situ Hybridization (FISH) in general (2)
- Uses fluorescent labelled ssDNA probes to hybridize with chromosomes immobilized on microscope slides to identify chromosomal aberrations.
- Gene-specific or locus-specific probes can be used to detect chromosomes
153
In FISH, when would a single-copy DNA probe be used?
Probe specific for a gene on a chromosome -> detects if gene is present
154
In FISH, when would a repetitive DNA probe be used?
A probe specific for centromere of a chromosome which will show the location of this given chromosome
155
In FISH, when would a chromosome "painting" probe be used?
Has many probes specific to different regions of the chromosome, "paints" the entire chromosome and can show where homologs are situated in the cell
156
Describe the Smith-Magenis sndrome detection by FISH (2)
- Centromere-hybridization probe for chromosome 17 shows 2 spots
- Probe that hybridizes to 17p shows 1 spot (due to heterozygous 17p deletion)
157
Describe spectral karyotyping (SKY) (2)
- Technique that all 24 probes (22 autosomes, 1 X chromosome and 1 Y chromosome) are combined and used for FISH of metaphase chromosomes
- Each chromosome-specific probe emits its own signal; thus, abnormal chromosomes consisting of pieces of different chromosomes can be identified (used to detect translocations)
158
Detecting mutations with subtle changes is difficult due to which 2 limitations?
1. Each cell has only 2 copies of a gene
2. Only a trace amount of genetic materials can be obtained from the cells
159
What are the 2 ways to amplify a DNA fragment of interest?
1. Fragment genome and PCR fragments
2. Do a restriction enzyme digestion and create a library of different fragments, which can then be isolated. Replicate the clone with the sequence of interest
160
What are the 2 types of libraries?
1. cDNA libraries: a combination of cDNA fragments inserted into a collection of host cells, which together constitute the "transcriptome" of the organism
2. Genomic DNA library: a collection of total genomic DNA from an organism
161
Difference between plasmid and vector
Plasmid: An extra-chromosomal DNA molecule found in bacteria naturally and can be replicated independently from the microorganism's own chromosomes
Vector: A plasmid that is engineered for the applications in molecular biology (e.g. bacterial artificial chromosome (BAC) vectors are designed plasmids containing large DNA inserts of 100-350 kb)
162
True or false: all plasmids are vectors, but not all vectors are plasmids
False
- All vectors are plasmids, but not all plasmids are vectors
163
Describe how DNA fragments are inserted into vectors
EcoRI endonuclease detects palindromic sequences: makes sticky ends, which allows for insertion of a DNA fragment with sticky ends
164
Definition of DNA library
A collection of clones, each of which carries a vector molecule into which a different fragment of DNA derived from the total DNA or RNA is inserted
165
complementary DNA (cDNA) is a (coding/non-coding) DNA fragment synthesized from an mRNA template used reverse transcription
Coding
166
In what stage are chromosomes that are analyzed by SKY?
Metaphase
167
Describe Comparative Genome Hybridization (CGH)
Used to determine the copy number (or dosage) differences between two distinct DNA samples
- Mix equal proportions of test (patient) DNA and control (normal) DNA that are dyed differently + hybridize into different wells (each well = predetermined region of the genome)
168
In CGH, greater fluorescence from test DNA indicates...
Duplication of given region in the test sample
169
In CGH, greater fluorescence from control DNA indicates...
Deletion of given region in the test sample
170
Difference between traditional CGH and array CGH (aCGH)?
Traditional method uses chromosomes fixed on a slide, while array is done on a microarray that may contain BAC clones
171
How does the traditional CGH work?
Overrepresented sequences will bind more easily to regions on the chromosomes so chromosomes will fluoresce with either test DNA colour or control DNA colour, based on which contains more of the sequence
172
When might aCGH be a superior method to traditional CGH?
If the deletion or duplication is very small, these changes are hard to see on chromosomes so a microarray maybe used.
173
Difference between DNA microarray and CGH microarray
DNA microarray uses probes fixed to a slide while CGH microarray uses free-floating probes (like BACs)
174
Whole-genome sequencing
Alignment of sequence reads from a patient's genome to the reference sequence
175
If alignment of sequence reads from a patient's genome to the reference sequence of 2 chromosomes reveals reads that contain contiguous (adjacent) sequences from both chromosomes, what does this indicate?
A translocation
176
What is mosaicism?
When a mutation occurs in one cell of the developing embryo, but not al of them
- Usually difficult to diagnose
177
What 2 things do unbalanced rearrangements result in?
Duplication and deletion
178
What is duplication usually caused by?
Unequal crossing over between misaligned homologous chromosomes or sister chromatids
179
Haploinsufficiency definition
The inability of a single copy of a gene to carry out the function normally performed by two copies
180
What results in Pallister-Killian syndrome?
Duplication of all or a portion of chromosome 12p
- Patients show characteristic craniofacial features, mental retardation and a range of othe rbirth defects likely to be related to trisomy or tetrasomy for specific genes present in the duplicated region
181
What is a marker (mar) chromosome/ supernumerary marker chromosome (SMC)?
- Type of unbalanced rearrangement
- Small piece of extra chromosome
- "in addition to normal chromosome complement
- prenatal frequency occurs at 1 in 2500
182
How are mars (SMCs) detected and why?
The mutation is difficult to be identified, often require chromosome painting by FISH (SKY)
183
mars/SMCs are usually present in what state?
Mosaicism
184
many marker chromosomes lack _____ and are likely to be ______
Telomeric sequences, small ring chromosomes (formed by two breaks and the broken ends of the chromosome reunite in a ring structure)
185
What is an isochromosome?
Results due to unbalanced arrangement, a chromosome in which one arm is missing (-> partial monosomy) and the other duplicated (-> partial trisomy) in a mirror-image fashion
186
What 3 diseases do isochromosomes cause?
Turner syndrome (one X chromosome of female partially or fully missing) solid tumours, and hematological malignant neoplasms
187
What are 2 possible mechanisms for isochromosome formation?
1. Mis-division through centromere (centromere divides horizontally instead of vertically)
2. Defective meiotic crossover (results in acentric chromosome and dicentric chromosomes, dicentric chromosome has both of the same arms)
188
Acentric chromosomes are degraded. Dicentric chromosomes are also degraded, unless one of which 2 things happens to form a pseudodicentric chromosome?
1. Dicentric chromosome is mitotically stable due to one of the two centromeres being inactivated
2. Both centromeres always coordinate their movement to one of the mitotic poles during segregation
189
What 2 techniques can be used to detect small deletions or duplications?
Multicolor FISH (SKY) and microarray
190
True or false: in general, balanced chromosomal rearrangements do not have a phenotypic effect for the carrier; however, it is critical to determine if the rearrangement is truly molecularly balanced because it may produce unbalanced/abnormal offspring (e.g. balanced X autosome translocation)
True
191
When does a chromosomal inversion occur?
When a single chromosome undergoes 2 breaks and is reconstituted with the segment between the break inverted
192
Paracentric inversion (does/does not) include the centromere and the breaks occur in (one/two) arms
Does not, one
193
Pericentric inversion (does/does not) include the centromere and the breaks occur in (one/two) arms
Does, two
194
Paracentric inversions can only be identified with which two techniques?
Banding or FISH using locus-specific probes
195
(para/peri)centric inversions are easier to be identified
- explain why
Peri
- Due to changes in banding patterns and the proportion of the chromosome arms
196
What 3 techniques may not be sufficient for detecting a pericentric inversion and why?
- G-banding may not be sufficient due to the presence of centromere
- CGH and microarrays can't be used because they don't detect structural info (and they're balanced so no duplication/deletion)
197
You have encountered a new genetic disorder that causes muscle weakness and heart failure in a newborn female baby. As a curious cytogeneticist, you went on to analyze the pedigree and found that the disorder is strictly maternally inherited in the family. Which on of the following approaches would be the most logical, efficient and cost effective for the next step of analysis?
a) Karyotyping by G banding
b) spectral karyotyping
c) analyzing nuclear DNA by array CGH
d) analyzing mitochondrial DNA by array CGH
e) whole-genome sequencing
D
198
What is the technical definition of mutation, and the human genetics definition of mutation?
Mutation: technically 'any change in DNA sequence"
In human genetics: any DNA changes that cause genetic disease; often affect only single genes; not microscopically observable
199
Polymorphism definition
Existence of multiple alleles of a gene in the population
200
Definition of a molecular disease and primary cause
A disease where pathology can be traced back to a single molecular factor
Primary cause: mutation, either inherited or acquired
201
Biochemical genetics definition
Studies the relationships between genes and the observable traits or phenotypes at the levels of protein production, protein function, biochemistry (eg. modifications), and metabolism (e.g. turnover)
- basically just looks at the relationship between genotype and phenotype
202
Describe splice-site mutations
Can have mutation at donor/acceptor sites -> spliceosome can't detect intron
- results in translated intron and altered protein structure
203
What can promoter mutations result in?
Inhibit binding of activated TFs
204
True or false: mobile element insertion (transposons) can cause mutations
True
205
Closer loci have a (higher/lower) chance of recombination
Lower
206
How is the genetic distance between two loci measured and what does this mean?
Measured in centimorgans (cM)
1 cM = 1% recombination frquency
207
Each hemoglobin contains which subunits?
2 alpha
2 beta
4 heme
208
Describe the active site of a heme unit
Has an Iron (Fe) ion, where O2 will attach
209
Cri du chat syndrome mutation
Deletion of 5p arm
210
DiGeorges syndrome mutation
Microdeletion on chromosome 22q11
211
Rett syndrome
Loss-of-function (under expression) mutations of the MECP2 gene, which encodes a nuclear protein that binds methylated DNA and recruits histone deacetylase to methylated DNA
212
MECP2 duplication syndrome
Gain of function (over expression) of MECP2 gene
213
True or false: A male with Down's syndrome has a triploidy
False; a male with Down's syndrome has a trisomy (21)
214
The Alu family is an example of what type of DNA sequence?
SINE
215
3 examples of specific transcription factors
Smad1, Smad2, beta-catenin
216
2 examples of basal or general transcription factors
TFIIA and TFIIB
217
What are 4 types of molecular factors that are critical in transcriptional regulation of protein coding genes?
1. Specific transcription factors
2. Enhancer/silencer regions
3. Basal/general transcription factors
4. RNA pol II
218
What is the first level of chromosome compaction in humans?
Involves DNA wrapping around histone proteins to form nucleosomes
219
True or false: The alpha and beta subunits in hemoglobin are encoded by different genes
True
220
What gene/chromosome are the alpha subunits of hemoglobin encoded by?
HBA gene on chromosome 16
221
What gene/chromosome are the beta subunits of hemoglobin encoded by?
HBB gene on chromosome 11
222
What mutation causes sickle cell disease?
Autosomal recessive disorder of hemoglobin where beta subunit genes have a missense mutation that substitutes Valine for Glutamic acid at amino acid 6 (G6V)
223
What effect does the G6V mutation in sickle cells have?
The G6V mutation in beta globin decreases the solubility of deoxygenated hemoglobin and causes it to form a sickle shape
- Normally, oxygenation of hemoglobin causes the hemoglobin polymer to dissolve/erythrocytes to regain its normal shape
- Sickle cells can still attach O2, but upon O2 attachment, they stay in polymer form which makes RBCs stiff
- Sickled erythrocytes clog capillaries because the sickle cells can't dissolve and squeeze through, which causes infarctions (i.e. tissue death)
224
What is hemolytic anemia?
Repeated sickling and unsickling of sickle cell patients produce irreversibly sickled cells that are removed from the circulation by the spleen
- Results in a lower number of RBCs in the blood
225
How is sickle cell anemia cured in general?
By replacing defective beta unit (adult) with normal gamma unit (fetal) in the RBC stem cells
- Genes that encode beta subunits are only expressed in adults. Fetuses express gamma subunit which have higher affinity for O2.
226
What protein acts as a molecular switch to change gamma subunit expression to beta subunit expression after birth?
BCL11A
227
What is the erythroid enhancer region's function?
When it's bound by BCL11A, it enhances beta subunit expression but silences gamma subunit expression
228
Describe how we can target RBC stem cells to cure sickle cell anemia
Use allelic editing to target the BCL11A enhancer in CD34+ cells (RBC stem cells)
- If BCL11A can't bind, then gamma will be expressed rather than beta
- These stem cells will be able to permanently take over in the bone marrow, and eliminate vaso-oclusive episodes and transfusion dependence
229
Over ____ genes associate with a single phenotype, which means...
3000
- No matter what type of disruption occurs to these genes, it will always result in the same phenotype
230
True or false: the number of genes with 1 phenotype are the highest, and this decreases as the number of phenotypes increases per gene
True
231
Mutations can cause disease through which 4 effects on protein function?
1. LOF of a protein
2. GOF of a protein
3. Acquisition of a novel property
4. Misexpression
232
Heterochronic expression of a protein
Expression of a gene at the wrong time
- e.g. beta subunit being expressed before birth
232
Ectopic expression of a protein
Expression of a gene in the wrong place
233
What is the most common mutation -> protein effect pathway?
- Provide an example
Mutations in coding region results in abnormal protein, leading to a LOF of the protein
- e.g beta-Thalassemias
234
What are 3 types of mutations that cause loss of function?
1. Nonsense mutation in coding sequence
2. Missense mutations in coding sequence
3. Mutations that cause protein instability, reducing the protein abundance (certain mutations can increase the degradation of proteins)
235
What can the severity of a disease be correlated with?
The degree/amount of function lost
236
Thalassemia is an example of a ____ mutation
LOF
237
Describe alpha-thalassemia
alpha-globin chains are deficient, so beta chains (or gamma chains fetus) are in excess, forming beta- or gamma- homotetramers, with greatly reduced oxygen-binding capacity (hypoxemia)
238
Describe beta-thalassemia
beta-globin chains are deficient, forming alpha-homotetramers that precipitate (i.e. form solid) and damage the cell membranes of RBC precursors. This results in premature erythrocyte destruction and anemia
239
What's the difference between thalassemia and sickle cell anemia?
Sickle cell disease has novel function (cells have high affinity for each other)
240
What are two examples of mutations that lead to beta thalassemia?
1. C to T (Gln36Stop) creates a nonsense mutation in beta-globin that leads to cause beta-thalassemia
2. Promoter mutations that decrease expression of the beta-globin mRNA to cause beta-thalaasemia
241
What is an example of a mutation that causes alpha-thalassemia?
Deletions of the alpha-globin genes
242
Monosomies, such as Turner syndrome, are examples of ___ mutations
LOF
243
Tumour suppressor mutations that lead to cancer are examples of _____ mutations
- Give an example of a cancer that is caused by this mutation
LOF
Example: retinoblastoma is caused by deletions in a tumour-suppressor gene RB1
244
What two things can GOF mutations result in?
1. Enhance the normal function of a protein
2. Increase production of a normal protein
245
Achondroplasia is an example of a ____ mutation, specifically which mutation?
GOF of FGFR3 that leads to short stature disorder
246
Trisomy 21 (Down's syndrome) is an example of a ___ mutation
GOF
247
Charcot-Marie-Tooth disease type 1A (CMT1A) is an example of a _____ mutation, specifically which mutation?
- Describe the disorder
GOF
- Duplication of PMP22 gene on chromosome 17; mutation leads to abnormal protein production and damages the myelin sheath.
248
Hb (Hemoglobin) Kepsey is an example of a ____ mutation , specifically which mutation?
- Describe the disorder
GOF
- Single amino acid substitution, beta-globin: Asp99Asn
- Disturbs heme-heme interactions at the alpha1/beta1 subunit interaction point
- High oxygen affinity, generally benign (doesn't cause disease)
249
What are novel property mutations in general?
A change in the amino acid residue can cause disease by conferring a novel property on the protein without altering its normal functions
250
Sickle cell disease is an example of a ____ mutation
- Describe why it's classified like this
Novel property mutation
- The novel property is that mutated globin chains aggregate to form polymeric fibers that deform red blood cells
251
Describe HBFH disease and its molecular basis for how this occurs
Expression of gamma globin in adults
- Normally, BCL11A binds the enhancer of the globin subunits, which then also binds the NuRD which recruits additional factors and acts as a silencer for the gamma globin gene. The LCR (like a second enhancer region) then binds co-activators and NF-Y to activate beta-globin transcription
- In HPFH, there is a BCL11A LOF, to NuRD is not being recruited to silence gamma-globin gene. LCR enhances expression of gamma-globin gene, and represses expression of beta-globin gene
252
What are the 8 steps at which mutations can disrupt the production of a normal protein?
1. Transcription
2. Translation
3. Polypeptide folding
4. Post-translational modification
5. Assembly of monomers into a holomeric protein
6. Subcellular localization of the polypeptide or the holomer
7. Cofactor or prosthetic group binding to the polypeptide
8. Function of a correctly folded, assembled, and localized protein produced in normal amounts
253
What are 2 classes of proteins based on their expression patterns?
1. Housekeeping proteins
2. Tissue-specific specialty proteins
254
Describe housekeeping proteins (3)
- Present in virtually every cell
- Fundamental for the maintenance of cell structure and function
- Account for 90% of the mRNAs expressed in humans
255
Tissue-specific specialty proteins
- Expressed in only one or a limited number of cell types
- Have unique functions
- Account for 10% of mRNAs expressed in human
256
Most clinical mutations are of (housekeeping/tissue-specific specialty) proteins
Tissue-specific
257
What 2 things can mutations in tissue-specific proteins cause? Provide an example of the latter
1. Disease restricted to that tissue (e.g. sickle cell disease)
2. Disease in a secondary site, where the protein is NOT expressed
- e.g. phenylketonuria (PKU) caused by the absence of phenylalanine hydroxylase in the liver; the brain (which does not express the enzyme), but not the liver, is damaged
258
True or false: no mutations in housekeeping proteins are lethal
False
- Although mutations in proteins rarely cause pathological changes, mutations in some housekeeping proteins (e.g. actin and DNA polymerase) are lethal
259
What 2 reasons explain why mutations in housekeeping proteins are limited to one or few tissues?
1. Genetic redundancy
- Other genes with overlapping activities reduce the impact of the LOF
2. The abundance of protein expressed
260
Genetic heterogeneity definition
A phenomenon in which a single phenotype or a genetic disorder is caused by mutations in a multiple number of alleles or loci
261
Pleiotropy definition
A phenomenon in which mutation of a single gene leads to multiple phenotypes or genetic disorders
262
Patau syndrome frequency
1/1000
263
Down syndrome frequency
1/700
264
Edwards' syndrome frequency
1/6000
265
What is the prevalence of autosomal disorders and unbalanced rearrangements at birth?
1/230
266
What is the prevalence of balanced rearrangements at birth?
1/500
267
Prenatal frequency of mar chromosomes
1/2500
