exam 1 Flashcards
1
Q
Genes can modify
A
MEAD metabolism excretion absorption distribution
2
Q
warfarin dosing
A
CYP (cytochrome P450) enzymes catalyze activation/inactivation of drugs
warfarin dose is genotype dependent based on type of CYP isoform
faster metabolism= high dose need
3
Q
Avery, Macleod, McCarty experiment
A
mice
heat killed virulent strain + rough non virulent strain = death
4
Q
nucleotide and uses (4)
A
nucleoside + phosphate group (1 or more)
constituents of DNA/RNA, cofactors, energy currency, cell signalling
5
Q
spontaneous deamination of bases
cytosine
adenine
guanine
5-methylcytosine
A
cytosine -> to uracil
adenine -> hypoxanthine
guanine -> xanthine
5-methylcytosine -> thymine
thymine cannot be deaminated
6
Q
analogs of base/nucleoside/nucleotide are used as
A
therapeutic agents
7
Q
base analog
A
5-fluorouracil (cancer therapeutic)
8
Q
nucleoside analogs (2)
A
gancyclovir (CMV retinitis therapy)
AZT/Zidovudine (HIV/AIDs)
9
Q
nucleotide analog
A
Adefovir (hepatitis)
10
Q
phosphodiester
A
5’ phosphage and 3’ OH bond of phosphate backbone
11
Q
forms of DNA
A
A- right handed, 11 bases/turn
B- predominant in humans, right handed, 10b/t
Z- left handed, 12 b/t
12
Q
DNA denaturation kinetics
A
zero order, NOT dependent on concentration
13
Q
DNA renaturation
A
2nd order kinetics, dependent on concentration
14
Q
purpose of non watson crick base pairing and examples
A
gene regulation and telomere stability
G-Quadruplex- G rick regions, increase telomere stability
i-Motif- C rich regions, pronated and neutral C on a single strand of dsDNA, cause stacking of DNA, vary with cell cycle phases (max in G1/S phase)
15
Q
alkylating agents
A
distort DNA structure
cyclophosamide, nitroureas, cisplatin
cistplatin bind to g -> intrastrand crosslinking -> distortion in DNA structure -> cell death
causes side effects because of indiscriminate binding
16
Q
start codon
A
AUG
17
Q
stop codons
A
UAA, UGA, UAG
18
Q
chromatin
A
organized structure of chromosomal DNA complexed with proteins
19
Q
nucleosome (core particle)
A
147 bp DNA
flat octomeric disc- made of histones (H2A, H2B, H3, H4)
20
Q
DNA charge
A
negative due to phosphate groups
21
Q
histone charge
A
arginine gives histones positve charge
22
Q
heterochromatin
A
transcriptionally inactive
higher methylation, low acetylation
present in mitosis, telemere, centromeric regions
23
Q
euchromatin
A
transcriptionally active
lower methylation, higher acetylation
present in interphase
24
Q
histones
A
102-135 AAs
conserved histone fold
N-terminal for modification (A)
3 a-helices connected by loops (B)
H3/4 most conserved across species
25
types of histone modification
- acetylation
- methylation
- phosphorylation
- methylation of CpG islands
26
acetylation
removes positive charge on histone (particularly on lysines) -> loosens interaction with DNA -> increases transcription
27
methylation
methylated lysine attract heterochromatin specific protein -> strengthen interaction -> gene silencing
28
phosphorylation
adds negative charge -> repel negative charge of backbone -> increases activity/ trancription
29
methylation of CpG
methylation of CpG island (promotor region) -> recruits methyl-binding proteines -> chromatin factors heterochromatin -> gene silenced
30
epigenetic regulation
control of gene expression by histone modification and modification of DNA bases but NOT the sequences (epigenetic- “on top of DNA modifications”)
31
PWS vs AS
| prader-willi syndrome and Angelman syndrome
deletions on chromosome 15q11-q13 causes 2 different disorders
maternal imprinted and paternal deleted -> PWS
paternal imprinted and maternal deleted -> AS
32
henderson-hasselbalch
pH= pKa + log [A-]/[HA]
pKa [A-} = [HA] for weak acids
33
pH> pKa
| pH
deprotonated
| protonated
34
amino group pKa
9.0
35
carboyxlate pKa
2.0
36
common buffers
used because most drugs are weak bases/acids, easier to pass membrane if uncharged
1. bicarb and carbonic anhydrase
2. Phosphate buffer
37
peptide formed through what rxn?
condensation
38
protein structure
1- AA sequence
2- linear arrangement alpha helix (intrachain H bonds) B-pleated sheets (H bonds between segments, parallel and anti-parallel, 4 AAs proline kinks and glycine H-bond formation minimal steric hindrance)
3- 3D conformation
4- subunit arrangements
39
disulfide bond
2 cysteines can stabilize protein fold
40
suprasecondary structures
beta-hairpin
coilied-coils
BaB
Greek key
41
huntington's disease
polyQ expansions (many CAG or Gln codon) lease to disruption in clathrin-mediated endocytosis -> cell death
42
prion disease
infected prion disrupts a helix and b pleated sheet to trigger abnormal protein folding in the brain
43
collagen structure and formation
rigid structure for support and structural integrity
3 left handed alpha helices form right handed triple helix, high pro and gly
formation:
1. hydroxylation
2. glycosylation
3. a helix formation
4. disulfide bond formation
5. triple helix formation
6. pretoeolytic cleavage
44
EDS
stretchy skin, inability to remove N/C termini or cross link alpha chains
45
osteogenesis imperfecta
brittle bones due to mutions that replace gly preventing triple helix formation
can be due to haploinsuffciency or dominant negative effect (loss of function becomes dominant)
46
elastin
found in lungs, walls of large arteries, elastic ligaments
47
marfan syndrome
mutations in fibrillin 1 (FBN1), leads to impaired structual integrity, autosomal dominant
48
elastase
degrades elastin in alveolar walls and other structural proteins
important for remodeling
49
alpha 1-antitrypsin
inflammation protection
serine protease inhibitor protects tissue from protease released by inflammatory tissues
50
smoking leading to emyphysema
smoking oxidizes methylation of a1-antitrypsin (inhibits elastase and other proteases), degrades elastin in lungs, damages lungs
51
bacteria vs humans DNA replication
Bacteria:
o Single origin of replication, initiated by dnaA
o Longer Okazaki fragments
o DNA Polymerase subunits:
▪ I: 5’-3’ polymerization, 5’-3’ exonuclease & 3’-5’ exonuclease
▪ II: 5’-3’ polymerization & 3-5’ exonuclease
▪ III (Replicase):5’-3’ polymerization & 3’-5’ exonuclease
▪ All DNA Pol have 3’-5’ exonuclease abilities, Pol I is unique in its addition of 5’-3’ exonuclease activity
▪ Pol IIIActually does most of the replication
o Use Topoisomerase II and IV
Humans:
o Multiple origins of replication, initiated by recognition complex
o DNA Polymerase subunits: α (primase), β (repair),γ (mitochondrial replication),δ (nuclear replication), and ε (nuclear replication)
o Use Topoisomerase I and II
52
helicase
(hexameric ATP dependent protein) unwinds the DNA
53
single stranded binding proteins
stabilize the unwound strands
54
initiation and opening
in bacteria:
1 replication of origin
bacterial initiator protein (dnaA)
in euks:
thousands origins of replications in humans
origin recognition complex binds to origin and denatures A-T base pairs
55
primer synthesis
in bacteria- primase
DNA polyerase III/replicase can add new nucleotides to the 3' strand
in humans- polymerase alpha makes RNA primers
DNA polyerase can add new nucleotides to 3' strand
56
removal of primers
bacteria- DNA pol 1
humans- RaseH
both have 5'-3' exonucleases
57
mutations in RNase H2=
neuroinflammatory disorder and SLE
58
DNA ligase (ATP independent)
joins fragments
59
type II topoisomerase (gyrase)
relieves supercoiling/ overwinding
cuts both strands allowing DNA to rotate
used in bacteria and humans
60
antibiotics and topoiomerase
quinolones/fluuroquinolones (antibiotics) target toposimoserase, prevent reversible ligation leading to cell death
point mutations in gyrase N terminal of GyrA and C term of Gyr B can lead to antibiotic resistance
61
Topotecan (chemotherapy) mechanism
used in ovarian and small cell lunger cancer
stabilizes the topo-I DNA complex and prevents the re-ligation, thus inhibits replication
62
end replication problem
for Euks
RNA primare cannot be placed for Okazaki fragment when the end of the DNA is reached (no room to clamp on)
section of the DNA will not be replicaticated (no polymerase available with 3'-5')
solution: telomeres- hexameric repeats of TTAGGGG (Tell them all genes get gone)
63
telomerase composition and activity
Composed of:
▪ Protein + RNA (Ribonucleoprotein→ RNP)
▪ Hexameric repeats (up to 15 Kb of repeating TTAGGG)
▪ Reverse transcriptase (RNA dependent DNA polymerase: hTERT)
▪ RNA, which functions as a template
▪ Short, single stranded regions that loop back to end and form G-Quadruplex (quartets) that stabilize the end of chromosomes
o Activity: Telomerase binds, extends 3’ end via RNA-templated DNA synthesis, f/b completion of lagging strand via DNA polymerase
▪ Telomeres also seal the ends of chromosomes to prevent undesirable fusion and prevent aberrant recombination and attach chromosomes to nuclear envelope
64
Telomeres in somatic cells, germ cells, stem cells, tumor cells, aging
```
somatic- no detectable telomerase activity
germ cells + activity
stem cells + activity
cancer cells + activity
aging + activity serves as mitotic clock
```
65
HIV and AZT
replication of HIV requires:
1 reverse transcriptase activity RNA to DNA
2 integrase activity incorporate into hose
AZT- analog of deoxythymidine, prevents chain elongation be reverse transcriptase (absence of 3 OH', prevents chain)
66
5-fluorouracil as a chemo agent
thymidylate synthase catalyzes synthesis of dTMP
dTMP -> dTTP (componant of DNA replication)
5-fluorouracil inhibits TS via substrate unavailability = no replication
67
DNA damage misincorporation
wrong base is placed/replication slips -> change overall code
68
DNA damage- deamination
base change
ex cytosine to uracil
69
DNA damage- depurination
removes purine from nucleotide, results in deletion of nucleotide pair
70
DNA damage- UV radiation
pyrimidine dimers, DNA cannot be copied
71
replication errors and repair
mismatch of bases or replication slippage (misalignment or repeat DNA sequences)
Repair:
DNA polymerase can repair through proof reading (3' to 5' exonuclease actvity)- mismatch repair
72
post replication errors
transitions- (purine to purine, pyrimidine to pyrimidine)
transversions- (pyrimidine to purine or vice versa)
frameshift- addition or deletion in bases, not a multiple of 3
structural changes- due to metabolic activity, heat, pH, radiation, or toxicity
spontancous changes- deaminations or dpurnations
UV damage
73
post replication repair
BER- base excision repair
| NER- nucleotide excision repair
74
how to fix replication error (mismatch repair) in bacteria
marks with methylation
MutS- sees mismatch, forms clamp
MutL- bind to MutS sliding clamp
MutS- activates MutH endonucleases
MutH nicks unmethylated strand
75
how to fix replication error (mismatch repair) in humans
single strand breaks or nicks provide signal repair
MutS- see mismatch
MutL- scans for nick
DNA and Exo 1 nuclease degrades strand
correct strand is repaired
76
lynch syndrome: hereditary non-polyposis colon cancer
mutations in MSH2 (homologue of metS) and MLH1 (humolog of mutL)
problems with detected areas to repair
77
BER
1. Wrong/damaged base is recognized and cleaved (Specific DNA glycosylase)
2. Sugar- P is removed (AP endonuclease and Phosphodiesterase)
3. Gap filled (DNA polymerase)
4. Nick sealed (DNA ligase)
78
NER
1. Either side of section is cut (Excision nuclease)
2. Section (oligonucleotide) removed (Helicase)
3. Repaired (DNA polymerase) and sealed (DNA ligase)
4. This pathway is either transcription coupled or follows a global repair pathway (both pathways involve a common set of proteins)
79
double strand break repair pathways (2)
homologous recombination- complete sequence restored, problems with this in BRCA
non-homologous end joining- sequence loss, altered segment with missing nucleotides
80
xeroderma pigmentosum
XP mutation
| skin malignancies
81
Cockayne sydnrome
ERCC6/ERCC8 mutation
| premature aging, photo sensitivity, hearing loss
82
colon cancer
hereditary due to mutations in repair genes
| MSH2 and MSH1
83
breast cancer
mutation in BRCA 1 and 2- defect in double strand break repair by homologous recombination
84
Werner syndrome
defect in BER
| cataract, short stature, premature graying
85
bloom syndrome
defect in double strand break repair
stunted growth, sunlight sensitivity, chromosome breakage, cancer risk
86
protein functions
```
signal structure
transport
energy product
immunity
enzyme
```
87
enzymes
specific type of protein
| all most all are proteins (ribozymes are made of RNA)
88
transferases
mediate group transfer (ex C,N,P)
89
hydrolase
cleavage of bonds via addition of water
90
lyase
cleavage of C-C, C-S, and C-N
tend to release CO2
form beta keto acids
91
isomerase
rearrangement
intramolecular group transfer
no net change in bonding, change formation
92
ligase
links 2 molecules, formation of bonds (C-O, C-S, C-N)
input of energy (ex ATP)
includes synthetases
93
reason for zymogens
inactive enzyme to prevent action in unwanted locations
protease enzymes are mase as zymogens then activated through hydrolysis
94
thiamine pyrophosphate (TPP), coenzyme
oxidative decarboylation, transfer of aldehyde
95
FAD/FADH2, coenzyme
redox rxn
96
NAD(P)+/NAD(P)H, coenzyme
redox e carrier
97
CoA-SH, coenzyme
acyl group transfer
98
Pyridoxal phosphate (PLP), coenzyme
transamination, deamination, decarboxylation
99
Biotin, coenzyme
carboxylation carries O2
100
tetrahydrofolate
transfers one carbon fragments
101
apoenzyme
inactive enzyme
102
holoenzyme
active enzyme
=apoenzyme + coenzyme
103
isoenzymes definition and example
proteins with same functional properties, but differences in sequence, produce diagnostic signatures
tissue damage-> large increases in cellular proteins
ex CK
brain B (pI= 5.34, negatively charged at pH 6)
heart M/B
skeletal M (pI=6.77, positively charged at pH 6)
can be seperated with native gel at particular pH
104
post translational modifications
1. phosphorylation
2. glycoslyation of extracellular proteins
3. ubiquitinoylation
4. Sumoylation
5. oxidation/reduction
6. acetylation
7. lipidation
8. methylation
105
post translational modifications, phosphorylation
control enzyme function, form salt bridges by recruiting target proteins, global regulation of cell function
ex- kinases add Ps
106
glycosylation of extracellular proteins
self recognition, accurate presentation to outside cell
107
ubiquitinoylation
makes proteins for degradation, targets lysine
108
SUMOylation
regulates protein regulates protein localization, protein-DNA binding, protein-protein interaction, transcriptional regulation, DNA repair
added to lysine to control other functions
109
ox/redox
facilitation disulfide bonds (made in oxidizing environments)
110
acetylation
affect selective gene transcription and chromatin function
| modifies interaction with other proteins, DNA (acetylated lys residues activate transcription)
111
lipidation
anchor modified proteins to different membrane locations, alters protein function
112
methylation
modify translation, alter protein structure, block protein modification particularly on lys or arg
113
michaelis menten assumes
1. steady state- [ES] is constant
2. free ligands- [S] >>> [E]
3. rapid equilibrium- kcat<
114
Vmax
maximum rate of rxn
115
Km
[substrate] at 1/2 Vmax
116
low Km=
high affinity
117
substrate regulation/ michaelis-menton enzymes curve
hyperbolic curve
118
allosteric regulation
binding of a molecule at regulatory site
sigmoidal curve
change rate smaller with change in substrate concentration
typically catalyze rxns with large decreases in free energy (large delta G)
binding of 1 molecule increases bindning of subsequent molecules (increases affinity) due to conformational change
119
ways to modify enzyme activity
substrate concentration
allosteric effectors/regulators (shift left or right)
post-translational modifications
abundance of enzyme
120
competitive inhibitors
binds to same place as substrate
vmax-same
Km- increases
EI only (no ESI)
121
competitive inhibitors examples
- statins on HMG-CoA reductase
- methotrexate blocks nucleotide biosynthesis inhibits dihydrofolate reducatse
- Salicylate cycloooxygenase (COX) block inflammation
122
noncompetitive inhibitors
binds to allosteric site
forms EI and ESI (non productive)
vmax-decreases
km- same
123
noncompetive inhibitors examples
cyandie bind cytochromes
D-JNKI-1 binds JNK block apoptosis of beta cells in pancreas
nifedipine- inhibits P450 CYP2C9, block morphine metabolism
124
mixed (uncompetitive) inhibitors
inhibitor binds to ES complex, making nonproductive ESI
rarer
vmax- decrease
km- decrease
125
mixed (uncompetitive) inhibitor examples
pepstatin- inhibits pepsin
trypsin inhibitor- inhibits trypsin
ethanol- inhibits acid phosphatase
126
irreversible inhibitor example
aspirin- covalently modifies COX 1 and 2 by acylation of ser near active site
```
advantage-
selectivity
lower doses
lower side effects
prolonged duration of inhibition
lower risk of drug resistance due to active site residue changes
```
127
common themes in physiology
1. structure determine function
2. homeostatic functions to maintain constant interval
3. cells can communicate/coordinate
4. substances/info moves across membranes
5. cell and body are compartmentalized
6. systems tend toward equilibrium, but many are kept at a steady state with energy input and output
128
homeostasis
body's ability to maintain a relatively stable (within narrow limits) internal enviornment
129
intracellular fluid
includes blood cells
130
extracellular fluid
vascular and interstitial fluid
includes plasma
131
can set points/intervals changes
yes
132
feedback system parts
```
stimulus
sensory receptor
afferent pathway
control center
efferent pathway
effector
```
133
negative feedback
return to set point to maintain homeostasis, most common
134
positive feedback
move further from set point, outside factor is required to shut off
ex child birth, blood clotting
135
autocrine
signal cell to target cell
136
paracrine
signal to adjacent cell
137
neurocrine
signal cell is a neuron, signal molecule is neurotransmitter
138
endocrine
signal to target cell over longer distance via hormone
139
canon's postulates
1. nervous system works to preserve conditions for organ function
2. tonic level of activity in many systems (blood vessels)
3. no tonic activity then antagonistic control (parasympathetic vs sympathetic)
4. same chemicals can have different effects (epi vasoconstricts intestines and dilates skeletal muscles)
140
growth
increase in cell number, size, extracellular matrix
141
differential growth
one part grows more than other
142
induction
chemical signal cause change in cells
143
cell differentiation
undifferentiated to differentiated cells
determination- activated
restriction- inactivated
metaplasia- de/re differentiation
144
selective cell death
cells programed to die
important for development
ex fingers and fetal brain
145
migration
movement of cells from one location to another
| amoeboid vs chemotaxis
146
epithelial folding
after inducation, edges of some types of undifferentiated, flat epithelia fold over themselves to form a tube or ball
147
cavitation/canalization
opening of paces in orginally solid tissues move to peripheral location
creates blastocyst cavity, celom and lumen of gut cavity
148
morphagens (4 examples)
diffusible molecules create gradients for developmental responses
TGF-beta
Bone morphogenic proteins (BMPs)
Hedgehog series
wingless related integration site (WNT)
149
TGF-beta
cell growth/differentiation, SMAD pathway
150
bone morphogenic proteins
cell differentiation
| pivotal developmental signaling molecules
151
hedgehog series
critical development gene
requires cholesterol to become active SHH
development of vertebrae
SHH-initiates paraxial mesoderm
muscle maturation
152
wingless-related integration sire
critical pattern development and axis pattern
muscle development/ maturation
differentiation of somites
153
notch signaling pathway
direct cell to cell contact
determine cell fate
delta like/jagged, transmembrane surface bound ligands interact with notch proteins
154
transcription factors
binds to DNA, regulate gene expression
```
includes:
histones
hox/homeobox
paried box (Pax)
basic helix loop helix (HLH)
```
155
receptor tyrosine kinases
cell surface receptors
involved in expression of growth factors
3 domains
extracellular ligand binding
transmembrane
intracellular kinase
156
meiosis
gamete formation
4n to 2n (23 replicated chromosomes) to n (23 nonreplicated chromosomes, 4 daughter cells)
1 DNA replication, 2 divisions
157
mitosis
4n -> 2n (2 daughter cells, 46 individual non-replicated chromosomes)
PMAT
158
gametogonium
first cell in line of gamete cell development, 46 replicated chromosomes, divides by standard mitosis, daughter cells can differentiated into primary gametocytes
159
primary gametocyte
46 chromosomes line up in homologous pairs (4n)
divides in division I (reduction division) to form 2 secondary gametocytes w/ 23 chromosomes (2n)
divides into divsion II to form 2 gamates (total 4) with 23 nonreplicated chromosomes (n)
160
spermatogenesis
```
spermatogonium (4n)
mitosis
primary spermatocyte
meiosis I
secondary spermatocyte (1n) x 2 cells
meiosis II
spermatid (1n) x 4 cells
differentiation
4 sperm
```
161
oogenesis
```
primary oocyte (46;xx)
meiosisI
first polar body (to get rid of extra DNA) and secondary oocyte (1n or 23;x)
meiosis II
3 more polar bodies, ovum (1n or 23;x)
```
162
heritability
proportion of the trait variation in a population explained by genetic favors
=0 genes do not contribute to disease
=1 genes fully contribute to trait/disease
163
locus
unique chromosomal location
164
allele
alternative forms of the same locus
165
codominant
codominant- both alleles are dominant ->additive effect on phenotype
166
haplotype
combination of alleles on the same chromosome
167
genotype
combination of alleles at a locus
can by homozygous or heterozygous
168
variants
difference in DNA between individuals
| most peoples genomes sequence is about ~99.5% indentical
169
polymorphism
MAF >1% frequency
does not imply effect on phenotype
170
mutation
MAF <1%
does not imply effect on phenotype
171
minor allele frequency (MAF)
the frequency of the least abundant allele in a population
172
allele frequency of A1
= # of A, alleles in population/ total # of alleles in population
173
main types of genetic variations
- single nucleotide variant (SNV)
- structural variation
- copy number variant (CNV)
- microsatellite
174
single nucleotide variant (SNV)
most abundant type of genetic variation
single nucleotide polymorphisms
most are bialleic but some have 3-4
source- DNA replication and repair
175
structural variation
can occur during DNA recombination
| involves more than 1 base bair
176
copy number variants (CNV)
DNA sequence whose number of copies varies between individuals
177
microsatelite
tandemly repeated sequences of 2-4 nucleotides
a type of copy number variation, but smaller than true CNV
178
germline mutation
mutation in every cell, heritable
179
somatic mutation
only some tissues/single cells have mutation, non heritable
| ex sporatic cancer and aging
180
mosaicism
the presence of cells with different genotypes
| result of somatic mutations
181
types of genetic variants
- neutral variants (most common)
- pathogenic variants
- functional variants
182
allelic effect size
alleles impact on disease
183
odds ratio
odds of disease in presence of allele/odds of disease w/o allele
>1 risk allele
=1 not associated with the disease
<1 protective allele
184
non-coding variants
- splicing (structural)
| - transcriptional regulatory region (ex enhancers/promoters)
185
intronic variants (2)
exon skipping
| intron retention
186
coding variants
silent
missense- 1 AA sub
nonsense- early term
frameshift- insertion/deletion, non multiple of 3
187
know naming
this replaced with this ter, count from change
188
loss of function variants
reduced or no function
most common
types: missense, nonsense, frameshift, splicing
recessive
189
gain of function variants
increased or new function
rare
types: missense, in-frame insertion, structure (gene fusion)
dominant
190
haploinsufficency
1 normal allele is insufficent for normal phenotype
LOF allele dominant
mild osteogenesis imperfecta (COLA1)
191
dominant negative (DN) effect
altered gene producted that antagonistcally affects the wt-gene
LOF alleles
missense mutations in COL1A1 leads to disruption in procollagen triple helix
severe osteogenesis imperfecta
192
monogenic disease
single strong variant drives phenotype
rare
```
examples:
muscular dystrophy
hutchinson-gilford
amyotrophic lateral sclerosis
CF
```
193
polygenic disease
multiple "weak" variants
common
```
examples:
obesity
DM
IBS
CVD
HTN
schizophrenia
```
194
susceptibility
sum of all genetic and enviornmental factors affecting disease
individuals above threshold have disease
195
association
tendency of 2 characters (ex alleles or disease) to occur to together at nonrandom frequencies. The strength of association can be measured by odds ratio
196
genes of monogenic disease can be indentified by:
linkage analysis
whole genome sequencing
whole exon sequencing
197
GWAS (genome-wide association study)
analysis of association between SNVs and disease
identifies alleles associated with the disease
analyzes 10,000 to 100,000 genes
can indentify new disease etiology and functions of different genes
198
polygenic risk score
composite of measure of genetic risk conferred by all disease-associated loci in an individual
determine how likely it is that an individual will get a disease
to find:
1. identify disease associated variants
2. in each individual add up effects of risk/protective alles to obtain individual PRS
3. correlate PRS disease risk in population
4. estimate individual relative disease risk
199
composition of chromosome
telomeres-GT rich sequences at the end of the chromosomes
centromeres
- metacentric: central
- submetacentric: intermediate
- acrocentric: terminal position
arms (p short, q long)-> regions-> bands
regions get longer, further down
200
```
abbreviation for
deletion
insertion
duplication
inversion
translocation
terminal
ring chromosome
isochromosome
```
```
del
ins
dup
inv
t
ter
r
i
```
201
karyotype and types (3)
the staining and display of chromosomes from metaphase spread
types:
G banding
FISH
Array CGH
202
g-banding karyotype
blood draw-> culture cells
lowest resolution
chromosomal imbalances, can detect aneuplodies, polyplodies, translocation, large deletions, inversions, duplication, isochromosome, ring chromosome
203
FISH
takes advantage of nucleic acid sequences and metaphase, looks at intact chromosome
looks for specific areas (centromeric, telmeric, chromosome-specific)
multiple FISH or SKY can look for more than one area at a time
benefits- medium resolution
can detect aneuplodies, polyplodies, translocation, small deletions, inversions, duplication, isochromosome, ring chromosome
limits- you have to know what you are looking for, specific location
204
Array CGH
patient compared to control DNA
green- increased pt DNA, red- more control DNA, yellow- equal
benefits-highest resolution, quicker
can detect aneuplodies, small deletions, duplication, isochromosome (not optimal), ring chromosome
used to detect intellectual disabilities in toddlers/newborns
limits-cannot detect changes not involving amount of DNA (inversion and balanced translocations), CANNOT detect triploidy or mitochrondrial DNA
205
translocations
Transfer of genetic material from one chromosome to another (non-homologous)
Can be balanced or unbalanced, reciprocal or Robertsonian
▪ Unbalanced: translocations with loss or gain of chromosome material
▪ Balanced: no loss or gain of chromosome material
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deletion
Loss of part of chromosome [del(chr#)(area deleted)]
o Can either be interstitial (w/in chromosome) or terminal (at end of chromosome)
o If deletions are large enough then it is not viable
o Large (visible with chromosome banding) or submicroscopic (microdeletions, detectable by FISH and aCGH)
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Inversions:
Reversing positions of chromosomal segments due to errors in replication/repair
o Can either be pericentric (involve centromere) or paracentric (don’t include centromere)
o Inversion carriers are asymptomatic (unless breakpoint is at important gene) but offspring can have clinical consequences b/c pairing/crossover is affected
o Only detects with chromosome banding or FISH (NOT aCGH)
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duplications
Extra copies of a segment of chromosome due to errors in replication/repair
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Isochromosomes
Loss of one arm with duplication of another
o Thought to occur due to centromere dividing transversely rather than longitudinally (with end up with having 2 p arms and no q arms and also 2 q arms with no p arms)
o Autosomal isochromosomes are lethal
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Ring Chromosomes:
Break occurs at each arm that fuse together
o Unstable in mitosis so its common to find ring chromosome in only a proportion of the pt cells. Other cells in individual are monosomic for that chromosome bc of absence/loss of ring chromosome
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euploid
normal chromosome number
2n somatic
n gametes
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ploidy
change in chromosome number
aneuploidy
polyploidy
213
changes in ploidy occur due to non-disjuntion in meiosis during gamete formation
meosis 1- all daughter cells affected
meosis 2- 1/2 daughter cells affected
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reciprical translocation
Breaking and exchange between chromosomes and formation of 2 new derivative chromosomes (can be balanced or unbalanced
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robertsonian translocation
Acrocentric chromosomes → New derivative chromosome
o Non- disjunction during meiosis can cause trisomies (e.g Trisomy 21)
can be determined by g banding or FISH
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autosomal dominant
: Expressed in homozygotes (AA) & heterozygotes (Aa)
o Parent to child transmission
o Every generation affected
o Unaffected parents do not transmit to children
o Males & females equally affected
o Male to male transmission (differentiates ADT from XLD
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Autosomal recessive:
Only expressed in homozygotes
o Unaffected parents can have affected children
o 25% of children affected
o Affected parents can have unaffected children
o Males & females equally affected
look for generation skipping
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x linked dominant
No male/female carriers → Those that have allele are affected
o Both males and females affected
o Mother transmits to daughters & sons
o Father transmits only to daughters
o Every generation affected
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x linked recessive
Males are always affected if they have disease allele
o Unaffected males do not transmit
o Carrier woman transmit to sons
o All daughters of affected males are carriers (Obligate carriers)
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consanguinity
mating occurs among relatives
homozygotes are more common if wide spread
suspect this in rare AR disease
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penetrance
Fraction of individuals w/ same genotype that show expected phenotype
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incomplete penetrance
Phenotype expressed in a fraction of individuals that all have the disease genotype
shown as percentage
ex: huntington's disease and retinoblastoma
223
Expressivity:
Range of phenotypes produced by a specific genotype
o Example: Marfan, Cystic Fibrosis, Polycystic Kidney Disease
224
Delayed Age of Onset:
Individual has genotype but does not develop condition until later in life
Example: Huntington’s Disease, most hereditary cancer syndromes
225
Pleiotropy:
Effects of a single gene on multiple organ systems/tissues
Example: Marfan, Cystic fibrosis
226
Locus heterogeneity:
Mutations in different loci produce same disorder
o Example: Retinitis pigmentosa, BRCA1/BRCA2
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Mutational heterogeneity:
Different mutations in same locus
o Example: Cystic Fibrosis, Beta thalassemia
▪ Individuals with two different mutations at the same locus → compound heterozygotes. Most individuals with AR disorders are compound heterozygotes unless their parents are related
228
new mutations
example achondroplasia
no family history
229
Uniparental disomy:
individual inherits both homologous chromosomes in a pair from a single parent. Can be isodisomy or heterodisomy
o Mechanism: Due to non-disjunction in Meiosis I or II in one parent
230
Unstable repeat expansion
Increase number of nucleotide repeats in successive generation
o If repeats exceed threshold #, disease occurs
o Mechanism: Slippage of DNA polymerase during replication in gametes or unequal crossover, repeats are usually CG rich
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Herdy-Weinberg Law
p+q=1
AA=p^2
Aa=2pq
aa=q^2
1/#
232
Hardy-Weinberg Assumptions
| 5
● Random mating
● No selection for any genotype
● No population migration
● Large population size
● No new mutations
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Expansion of trinucleotide repeats:
increase in number of repeats in successive generations
Can be transmitted as autosomal dominant, autosomal recessive, or X-linked
234
anticipation
Progressively earlier age of onset & severity of symptoms due to increased number of repeats
235
Law of independent assortment
members of different gene pairs assort to gametes independently of one another
o Not true for genes on the same chromosome they will be inherited together (in genetic linkage)
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Recombination:
occurs between homologous chromosomes during meiosis I… when 2 loci are on chromosome, they can be separated by recombination
o when 2 loci are 1 cM apart crossover occurs 1/100 meioses
o recombination is more frequent in female than in male games
237
haplotype:
combination of alleles at linked loci on chromosome (new haplotypes different than parental) are found in gametes
238
genetic linkage analysis
pattern of makers in affected individuals
pros:
determines genomic interval where disease lies
cons:
slow
requires large families with disease
requires additional method to find actual gene and causative mutations
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whole genome sequencing
next generation sequencing of whole genome
pros- few affect family members or relatively fuew unrelated affected individuals
good for monogenic disease and new genes
cons- expensive
privacy impacts
240
whole exon sequencing
same as WGS but misses
| intronic, regulatory, and non-coding variants
241
methods used for identification of monogenetic disorders
linkage analysis
| whole genome and exon sequencing
242
methods used for testing/screening
```
PCR
PCR-RFLP
ARMS-PCR
Allele-specific oligonucleotide hybridization
Southern blotting
Sanger Sequencing
```
243
gene expression methods (RNA)
nothern blots
| gene expression microarrays
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PCR
amplification of area of interest
can detect:
- insertions
- point mutations
- viral and bacterial infections
very sensitive, requires little DNA
245
PCR-RFLP
| restriction fragment length polymorphism
detects point mutations
smaller fragments if mutation is present
246
amplification-refractory mutation system PCF
ARMS PCR
allele specific primers
o Wt-specific primer only extends when annealed to Wt DNA
o When annealed to Mut DNA Mismatch @ 3’ end and no product
o 2 separate PCR reactions per pt sample
Amplification product with specific primer pair will only be obtained if there is no mismatch
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Allele-Specific Olgionucleotide (ASO) Hybridization
Similar to ARMS-PCR, uses allele specific oligonucleotides to hybridize only Wild type (Wt) or Mutant (Mt) allele
Utility
▪ Detection of point mutations, small deletions (bps), & small insertions
▪ Doesn’t require electrophoresis
▪ Can test for several mutations at a time (multiplex
248
southern blotting
detects insertions, deletion, point mutation that disrupt restriction site
good for trinucleotide expansions
no amplification required
radiolabed probe, size electrophoresis
249
sanger DNA sequencing
"gold standard"
pros: picks up all mutations within region
do not have to know what you are looking for (good for unknown mutations)
high fidelity
optimal for many mutations on same gene
indentifies deletions, insertions, duplications, point mutations
cons: expensive
time consuming
250
detect changes in gene expression
northern blotting
gene expression microarrays (red, yellow, green)
251
tissues to test
must test effected tissue where gene is expressed
252
gene therapy in vivo
WT gene delivered to the patient (ex CF)
253
gene therapy ex vivo
target cell removed and vector introduced
much lower risk of immune rejection
254
viral vectors
remove disease causing aspects of virus (infect but to lyse), just for delivery (vector and packaging)
pro:
more effective
con:
can elicit immune response or inactivate essential gene
255
non-viral vectors
less efficient, lower risks, assemble lysosome for delivery
256
CRISPR
Edits DNA (cut and paste), for cell response memory, not clinical yet
