CMMB final material Flashcards
The human genome main
- two copies in every somatic cell
- one copy in gametes
- 22 autosomes and 2 sex chromosomes
Genetic variation in normal populations
a) chromosomal level
b) CNV/person
c) single nucleotide level
Genetic variation also causes disease
a) whole chromosome (aneuploidy) Trisomy 21 (Down’s Syndrome)
b) Partial chromosome imbalance: seen by FISH
c) single nucleotide substitutions= Achondroplasia (little people)
polymorphism
any change within a popualtion thats >1%
-rare variant- anything less than 1%
Types of DNA polymorphisms
- single nucleotide insertion/deletion
- microsatellite
- minisatellite
- copy number variant (CNV)
- single nucleotide variant (SNV)
single nucleotide insertion/deletion
- many in the genome, unstable through generations
- unstable because DNA reproduces a second strand that can slip causing gain/lost od nucleotides
Microsatellites
- repeat units are 2-5 nucleotides in length
- also called Short Tandem Repeat (STR)
- certain number of repeats= many alleles in normal populations
Microsatellite uses
- DNA fingerprinting (everyone has different number of repeats)
- also half are passed off to kids
- disease: finding where those genes are located
Minisatellites
- repeat units are 10-100 nucleotides in length
- also called VNTR–> variable number of tandem repeat units
- Southern blot used
Copy Number Variation
- deletions and duplications
- range from 200 to >2000000 nucleotides
- usually 0-4 copies
Single nucleotide variants (SNVs)
- most common type of sequence variation
- more than 3000000/person
- many different effects: benign to disease-causing
Origins of Sequence Variation/Mutations
- replication errors
- DNA damage (deamination, depurination, demethylation) also by mutagens
Large deletion/duplication mechanisms
- more likely to happen in repetitive sequences
- if homologous sequences are very similar wrong ones can align together
CMT1a
Charcot-Marie-Tooth
- duplication
- progressive peripheral motor and sensory neuropathy (numbness/weakness)
HNPP
- Hereditary Neuropathy with Liability to Pressure Palsies
- peripheral nerves are unusally sensitive to pressure and results in numbness, tingling, and/or loss of muscle function
-deletion
Haemophilia A
- inverted repeats
- found in 45% of patients
normal is
1-21,22,23
affected
22, 21-1, 23
Translation refresher
a codon= 2 base pairs (total=64 codons)
-each codon= 1 amino acid
20AA
3 STOP: UGA, UAA, UAG
1 START: AUG (met)
Nomenclature (DNA level)
nucleotide #1: A in ATG (start)
upstream nucleotides are negative eg) 2 bps to the left= -2
downstream coding nucleotides numbered normally–> introns are skipped
amino acids numbered starting with the start codon=1
Mutation Types
A) Nucleotide Substitutions -AA effect: synonymous (silent) non synonymous (missense) nonsense splicing effects
B) duplication/deletion
- small
- large (one or more exons)
C) dynamic mutations
Synonymous (silent)
-DNA sequence change doesnt change the actual AA that is coded
Non-synonymous (missense)
-DNA sequence change; CHANGES the AA
Nonsense
premature stop codon
-ie) CTA–> CTG both are LEU
if this is found as the 325th AA in a protein how would you write that?
p.Leu325Leu
CTG–>CCG (LEU–> PRO)
if this as the 112th AA in a protein how would you write that?
p.Leu112Pro
TTA–> TAA Leu–> Stop
if this is found as the 33rd AA in a protein how would you write it?
p.Leu33*
Read-through (nonstop)
TGA–> GAA stop–> GLY
Mutated start codon
DNA seq changes the AA thats coded
ATG–> GTG Met–> VAL
p.Met1Val
this is the disease causing mutation in BRCA1
Splicing
The noncoding (introns) are removed from the main transcript= mature mRNA
Cuts after before GT of the intron until after the AG end of the intron
What happens when a splice site is disrupted?
Exon can be skipped
It’s almost impossible to predict what might happen
Intron might not get spliced out= keep going until the next one is found= extra DNA sequence
Small duplications/deletions
- 1 or more nucleotides can be inserted/deleted/ both
- if the number of bases are not multiples of 3= then there’s a shift of the codon reading frame (frameshift mutation) = Nager Syndrome
Large duplication/deletions
- 1 or more exons are duplicated (rare) or deleted
- most common is x-linked duchenne muscular dystrophy (70% of patients have a duplication/deletion of the DMD gene)
.33 of patients have a de novo mutation (not found in the mom)
Autosomal dominant
All generations are affected but not all affected have an affected parent; maybe low penetrance?
Autosomal recessive
All parents would have have to be carriers; would have to have a very high carrier frequency for 3 members of the family to each marry a carrier
X-linked dominant
Not all affected have an affected parent; low penetrance
X-linked recessive
Unaffected father with affected grandsons; affected female
Dynamic mutations repeat expansions give an example
Fragile X syndrome- caused by repeated (CGG)n near the promoter of the FMR1 gene
Normal= 6-54
Permutation=55-200
Full mutation= 200- 1000
Permutation carrier female= affected offspring
Permutation carrier male= permutation carrier offspring and affected grandsons from his daughter
Risk of expansion depends on the size of the permutation
Dynamic mutations
A major cause of neurological disorders
Expansion of a simple repeat in coding region or noncoding region
Usually demonstrate reduced penetrance alleles and variable expressivity
Consequences of mutation
Loss of function= Fragile X
Friedreich Ataxia
Gain of function= Huntington (coding), Myotonic Dystrophy
Anticipation
Earlier onset
Size of the expansion proportional to age of onset
Bigger the expansion the more unstable it becomes for the next generation and earlier age of onset
Myotonic Dystrophy Type1
CTG repeat in the 3’ UTR of the DMPK gene
Mild 50-150: cataract, mild myotonia
Classic 100-1000: muscle weakness and wasting, myotonia, cataract, cardiac conduction abnormalities
Congenital: >1000 hypotonia/severe generalized weakness at birth, respiratory insufficiency (early death)
Anticipation through maternal inheritance
Myotonic dystrophy
Anticipation inheritance
Huntington’s
Outcomes of the post HGP era
What do each of is carry
76-190 rare non-synonymous changes that are predicted to be deleterious
<20 loss of function and known disease-associated mutations
40-80 SNPs that are not present in our parents (new seq changes)
Variant Nomenclature
cDNA (RNA) level
duplication
insertion
protein level
all numbering is in relation to the A in ATG (start codon) increases from there on
- duplication: 1 or more extra bases are present that are identical to those preceding it
insertion: 1 or more extra bases prsants that DO NOT match this preceding it - protein level- all AA are numbered starting from MET
Loss of Function Mutations
protein function is lost or reduced in the cell
- can result from missense, splicing, frameshift, or large deletion/duplication
- usually recessive mutations (both copies are mutated)
Cystic fibrosis- loss of function mutations in the CFTR gene
Gain of function mutations
- mutations that enhance normal protein function
- usually dominant disorders
ex) Achondroplasia–> single nonsynomous protein
- binds all the time doesn’t let the bones grow
- FGFR3
Novel Function
-proteins gain a new function or property
Sickle cell anemia:
RBC become sickled when deoxygenated and eventually stay sickled
-organ damage(liver) anemia and recurrent infections
also Huntington’s disease: presence of an expansion gives the genes new function–> neurone slowly killed
Dominant Negative Mutations
- a mutation that results in a protein that adversely affects the normal product within the same cell
ie) Osteogenesis perfecta (nonsense frameshift ) –> having abnormal collagen is worse than no collagen at all
Heterochronic/Ectopic expression
- gene is expressed at the wrong time (heterochronic) or in the wrong place ectopic–> often dominant disorders
- deletion in the beta-globin locus for fetal hemoglobin–> where the gene isn’t turned off when it should be
haploinsufficiency
- individual that is heterozygous for a certain gene is clinically affected because 1 gene is not enough for normal function
- if mutation is from normal parents= its a de novo mutation
- inherited as dominant disorders, with variable expressivitiy (can be inherited or de novo)
Gene dosage
- extra copies of normal gene products are sufficient to cause disease
- inherited as dominant disorders
- may have variable penetrance and expressivity
ie) Charcot-Marie-Tooth Disease Type 1A
- extra copy of the PMP22 gene where the loss can also cause disease
Mendels law of segregation
-every individual has 2 alleles for each trait–> one will be randomly passed on to offspring
Mendel’s Law of independent assortment
-separate genes for separate traits are passed independently to offspring
gene linkage
certain genes usually inherited together, because they are on the same chromosome. Thus parental combinations of characters are found more frequently in offspring than non parental. (parental vs recombination)
genetic marker
A gene or (a fragment of) DNA sequence having a known location on a chromosome, has an easily identifiable phenotype and whose inheritance pattern can be followed.
allele
one of the alternate versions of a DNA sequence at a given marker
law of independent assortment
- linked
- unlinked
linked: one allele from Parent 1 is associated with one allele from Parent 2 >50%
unlinked:
disease state will be found equally associated with both marker alleles
Genetic Maps
- distance between markers is the recombination rate (theta)
- where 1% recombination= 1cM
- framework for linkage mapping
LOD formula
log10 x (prob of birth seq with a given linkage value/ prob of birth sequence with no linkage) = log10 (1-0)^NR x0^R )/(0.5 ^(NR+R) 0=THETHA
LOD SCORE
- stat test used for linkage analysis
- in order to find out if the two loci are actually linked or the data was linked totally by chance
- positive= linkage (greater than 3)
- negative= no linkage
uses of genetic linkage
- used by clinics before DNA sequencing was available to determine risk of being carrier, affected etc
- positional cloning: identify the genomic location of a disease gene without any prior knowledge on where or what the causative gene is.
- both are family context-dependent
What is cancer?
Clonal multistep process of the genes involved in growth
-uncontrolled growth
Neoplasia
New growth
Hyperplasia
Too much growth
Dysphasia
Incorrect growth
Benign
Localized growth
Malignant
Capable of growth
Metastasis
Distant growth
Cancer is a multistep process
Not always a mutations; can be caused due to a gain or loss of genes; every cancer is different with different genes involved
-accumulation of genetic errors in genes that control growth
First step can be inherited=cancer in families (some can be DNA repair genes)
Inheritance of Retinoblastoma
Eye tumor in childhood but not always
Sporadic inheritance: just have one individual no one else in the family has it
Familial inheritance: in every generation or skips generations
Mendelian
5% inherited
-in the germline is inherited; lets say you visit some place you increase the chances of having a somatic mutation–> earlier onset
affected individuals in every generation of a pedigree (can skip generations as well)
Sporadic
95% sporadic
-waiting for random exposures to knock out genes=cancer
dying old old age= accumulation of mutations over time
only 1 person affected in a pedigree
Familial Adenomatous Polyposis
- multiple polyps (colon cancer in young adults)
- inherited mutation in gene that regulates cell division in colon
- APC gene mutation
- Mendelian
Li Fraumeni Pedigree
- multiple types of cancers
- mendelian
- mutation in a gene that regulates cell replication in multiple cell types
TP53 gene mutation
**if theres a legend=autosomal dominant
Knudson’s 2 Hit Hypothesis
1st hit= inherited
2nd hit= acquired
Inherited Cancer Predisposition
-inheritance of genetic alteration, normal phenotypes at birth
RB1 retinoblastoma
APC- Familial Adenomatous Polyposis
TP53-> Li Fraumeni pedigree
inheritance of genetic alteration, part of a recognizable syndrome
Down Syndrome
Chomosome instability syndromes
chromosomes are broken because DNA repair mechanisms dont work, therefore accumulating errors
(basically mutation in genes responsible for DNA repair
Faconi Anemia
Bloom Syndrome
Ataxia telangiectasia
Xerderma pigmentosum
Down Syndrome
-trisomy 21–> tongue is out because mouth is too small; gap btw big toes and the rest of the toes;
High risk of transient myelodyplastic syndrome at birth (cells dont look right; sometimes can b acute; individual gets older quicker (avg age 32)
10-100 fold risk of acute megakaryoblastic leukemia –> leukemia that affects platelattes in the blood
Fanconi Anemia
growth retardation (thumb sticking out)
thumb anomalies
increased risk of of leukemia and liver cancer
chemical induced chromosome breakage –> unrepairable
defect in DNA repair enzyme
Bloom Syndrome
- facial butterfly rash
- more in the Jewish
- increased risk of leukemia
- increased sister chromatid exchange –> Harlarlequin banding)
mutation in BLM gene–> 1 chromosome completely light other one completely dark; normal you might have one maybe 2–> these individuals have many of these chromosomes
DNA unwinding enzyme:
-cant correct; cnat make the the DNA into RNA -> protein
Ataxia Telangiectasia
- occular telangectasias (corner of eyes have fine blood vessels)
- loss of muscle control (not being able to control walking
- lymphoid mailignancies (will not take body xrays etc)
mutation in the ATM gene
DNA repair enzyme
Xeroderma Pigmentosum
extreme sun sensitivity
blistering and skin cancer
mutation in UV dimer repair enzyme
Environmental Causes of cancer progression? LIST
radiation chemicals viruses diet lifestyle
radiation
- UV light from the SUN= melanoma–> cancer of the pigment in skin (depth not size that matters)
- ioniaing radiation in an atomic bomb= Leukemia
- previouslt X-rays caused lung cancer
Chemicals
asbestos–> lung cancer
smoke soot (small boys used to clean chimneys) scrotal cancer
fertilizers, pesticides= plasma cell neoplasms
Viral
Hepatitis–> liver cancer
EBV–> Burkitt’s lumphoma and others
HPV-> cervical cancer
HIV–> primary effusion lymphoma and others
Lifestyle
smoking= lung cancer
prostitutes: cervical cancer
nuns= breast cancer because nit getting pregnant etc
diet
nitriles in preservatives= liver cancer
- alcohol= liver cancer
- lack of fibre–> colon cancer
Japanese graph
stomach cancer is higher in the Japanese compared to the California caucasians whereas prostate cancer is higher in the the California Caucasians than the Japanese
Cancer is a Genetic Process
- accumulation of defects in genes involved in cell replication and cell death
- grouped by function
Cell replication Life cycle
1) oncogenes= green light
2) grows
3) splits into 2 cell
4) TSG gatekeeper genes= STOP
5) cell is repaired (TSG) caretaker genes
6) and the cycle starts again
Tumor suppressor genes
LIST GATEKEEPERS AND CARETAKERS
GATEKEEPERS
-RB1 and TP53 both are cell cycle regulators
CARETAKERS
- BRCA1 (repair DNA double)
- BRCA2( strand breaks)
- MLH1(repair DNA mucleotide)
- MSH2(mismatch)
Gatekeeper TSG: RB1
-eye tumour caused by hits in each of the 2 copies of the RB1 gene that suppresses cell growth within the eye
Familial: 1st hit inherited 2nd acquired (mendelian)
Sporadic: both hits are acquired
La Fraumeni pedigree
TP53 gene mutation
tumor supressor gene
gatekeeper of cell cycle
multiple cell types
Gatekeeper TSG: TP53
- checks DNA for damaged prior to making a copy of it
- if the checkpoint is non-functional
- leads to accumulations of mutations
- affects many cell types
DNA damage (radiation)–> cell cycle is arrested–> irreplaceable damage–> APOPTOSIS (elimination of damaged and a potentially cancerous cells)
OR
DNA damage (radiation)–> cell cycle arrest–> DNA repair
Caretaker TSG: MLH1 & MSH2
- repairs DNA replication mistakes (spell check)
- non-function leads to accumulation of genetic mutations
- inheritance of the MLH1 & MSH2 mutations
- hereditary Colon cancer (Non-Polyposis type)
Function of Cancer genes
oncogenes–>TSG’S
1) Gatekeeper–>AD
2) Caretaker–> AD and AR
=chromosome instability syndromes
Function of Oncogenes
- genes involved in cell proliferation
- WT= c-onc (cell oncogene)
- carcinogenic when upregulated
- mutated type= onc
What year did the first association of a chromosome rearrangement with cancer?
-example
-Chronic Myeloid leukemia caused by the philadelphia chromosome (extremely tiny)
1960
what year was the human chromosome G-banded
1970s
Chromosome Rearrangements
a) inversion
b) insertion
c) translocation
inversion
- chromosomal rearrangement
- chromosome is reversed from end to end
centromere included= pericentric
not= paracentric
insertion
-DNA segment from 1 chromosome is inserted into another chromosome
translocation
- transfer a segment of one chromosome to another chromosome
- if 2 nonhomo chromosomes exchange parts that the transloaction in reciprocal
Robertsonian translocation
translocation btw 2 acrocentric chromosomes by fusion at or near the centromere, with the loss of the short arms
FISH
1990s
-denature–> hybridize with a fluorescent probe–> visualize
Gene Fusion found by FISH
Philadelphia Chromosome Rearrangement
------------ ABL 9 gene //////////// 22BCR gene
———///// ABL/BCR gene t(9;22)
-precise rearrangement= creates a fusion gene and a fusion protein
ABL1= oncogene BCR= breakpoint cluster region
Fusion Protein Targetted Therapy
ABL1-BCR gene fusion responds to tyrosine kinase inhibitors
-drug is specially made to inhibit the ABL1-BCR fusion protein (CML/AML/ALL)
2008 WHO Classification
- WHO classifications of tumours are based on the primary genetic rearrangements
- dont need to know what it is to remove it, but do need to know what it is to treat it
Genes in Cancer Diagnosis: Fatty tumors
- chromosomes 3; 12
- lipoma
- atypical lipomatous tissue/well differentiated lipoma or dedifferentiated lipoma
- myxoid liposarcoma
Genes in prognosis: neuroblastoma
- neuroblastoma
- childhood solid tumour
- in nervous system
- outside of the brain
ALK FISH predicts Crizotinib Response
- NSCLC: non-small cell adenocarcinoma of the lung
- ALK FISH rearrangements in 3-5%
- Crizotinib significantly reduces tumor burden
ALK ATP binding pocket –> ALK in Personalized Medicine
Crizotinib aka XALKORI
Phase 2: 79/82 substantial reduction in tumor burden
Phase 3: FDA approval in 36 months
Alberta Thoracic Oncology Program
EGFR mutation positive:–> Erlotinib/Gefitinib
ALK rearrangement positive: crizotinib
Genetics
the study of hereditary and the variation of inherited characteristics
Epigenetic
-heritable changes in gene expression not caused by changes in DNA sequence
Waddinton’s Epigenetic Landscape
the epigenome tells the cell what to be
Molecular mechanics that mediate epigenetic
RNA/histone modification/ DNA methylation all cause Chromatin (euchromatin or heterochromatin) remodelling/heritable gene expression
*gene expression of daughter cells is the same as the mother cell
epigenetic phenomenon
- x-chromosome inactivation
- genomic imprinting
- centromere/telomere function
etc
euchromatin
-active
-gene rich regions
transcription occurs
-less condensed
interphase chromatin
- highly condensed
- gene poor
- found in transcriptionally inert regions
chromatin’s role with histones
DNA wraps around the histone twice and nucleosomes compact it
what are the basic building blocks of Chromatin
histones
-dimers form tetramer which form histone octamer (DNA of tetramers wrap around twice)
-the histone tails interact with DNA and these tails serve as the basis for the epigenetic marks
Post translational modifications of histone tails
- phosphorylation
- ubiquitination
- acetylation
- methylation
Features of Histone Modifications
- covalently attached groups to histone tails
- methyl
- acetyl
- phospho
- ubiquitin
- reversibile and dynamic
- directs transcriptional signals -> DNA is negatively charged and histone tail is positively charged–> if you take aways the positive charge= they will no longer interact anymore
Histone modifiers
a) Writing
acetylases/methylases/phosphorylases (add groups)
b) Erasing
deacetylases, demethylases/ phosphatases
-epigenetic marks need to be erased for genes to be expressed
c) Reading= chromatin remodelling factors
- interpret the marks (remodel the chromosome)
Chromatin and Nucleosomes
nucleosome moves or is physically removed by the chromatin remodelling to gain or inhibit access to the promoter–> can also condense or add nucleosomes
-nucleosome spacing/mobility/ assembly
histone acetylation is associated with gene activation
inactive= no acetyl groups active= acetyl groups attached
- transcription factors tell DNA to open (acetylate)
- you cant change DNA but you can change the acetyl marks
ChIP
Techniques to study Histone Modifications
-Chromatin immunoprecipitation: Strategy for localizing histone marks
ChIP-qPCR
if you know the target gene
ChIP-seq
or other genome-wide techniques: unbiased
limitations of ChIP
a) antibody specificity: need 1 that corresponds to what you are looking for
b) inherent biases of localization methods
techniques to study histone modifications
- take DNA and crosslink then fragment and then put an antibody in it and the protein of interest and DNA attached will bind to the antibody
- remove the proteins and you are left with the DNA you need. do massive sequencing of your DNA the more DNA your antibody pulls down, the more you sequence
ChIP vocab
- depth
- coverage
- the number of mapped sequence tags
- how much of the genome the reads can be mapped to
if a gene is turned on, why would you have repressive marks?
-to prevent criptic transcription (noncoding RNA) or to repress isoforms (can alter gene function)
inactive genes are full of
repressive marks
the histone code hypothesis
- you can predict if a gene is on or off, but its more complex
- there are many different factors that influence the genome
Histone cross talk
basically DNA methylation will try to turn off transcription and another might try to turn it on (interference with each other)
DNA methylation
- chemical modification of DNA
- can be inherited without a sequence change
methylation most frequent at
5’CpG 3’ dinucleotides
CpG islands
(methylation(
-regions of high CG content 60% of mammal promoters found here
DNA hypermethylation=
associated with gene silencing
promoter unmethylated
genes can be transcribed
promoter methylated
gene is silenced
methylated binding proteins recognize CpG island promoters then
either methylation or histone modification causes methylation to occur
x chromosome inactivation
males: y chromosome lost a lot of ancestral genes
females: silence most genes on 1 x-chromosome
barr body
x-inactivation proof
-properties of X inactivation (late replication in the S phase)
-remains condensed in interphase
XIST
- interacts with proteins which interact with histones and methylation of histones of soon to be inactive x happens
- once x is inactive, it can’t become active
genomic imprinting
zone you inherited something from
-genes that are not turned on are methylated
-differential expression of genes depending on the parent-of-origin
disorders of genomic imprinting (2)
Prader-willi Syndrome (dad) and Angelman Syndrome (mom)
-both are caused by the same deletion= determined which parent they inherit it from
epigenetics and cancer
- you can’t change the genome and cancer occurs in the genome
- all cancers start with DNA (usually a mutation)
- then a gene is shut off and this causes changes within a cell
- stem cell looks like a cancer cell in the epigenetic level
treatment of cancer by modifying the epigenome
- mechanism of action
- direct incorp into DNA
- blocks effects of DNA methyltransferase
- causing hypomethylation of DNA
- reverses inactivations of tumor suppressor genes
- can lead to cytotoxicity
cytosine analog–> insert DNA into the helix therefore unmethylated cytosine, now tumor expression (or w/e gene) turns on
ENCODE what it stands for?
ENCyclopedia Of Dna Elements
obj of ENCODE
find all functional elements
- bound by specific proteins
- -histone modifications/DNA mthylation/transcribed(epigenetic features)
ENCODE use obj too
genes (coding and noncoding) promoters enhancers (activation/silencing) specific transcription factor binding sites insulators chromatin states
ENCODE methods
RNA-Seq
different fractions of RNA–> sequencing
ENCODE methods
ChIP-seq
chromatin immunoprecipitation- DNA binding protein–> seq
ENCODE methods
DNase-seq
nucleosome- depleted DNA–> seq
ENCODE methods
RRBS
bisulphite treatment–> unmethylated C—->U–> seq
ENCODE methods 3C,5C
chromatin interactions–> sequencing
5C=Carbon Copy Chromosome Conformation Capture
- crosslinking
- digestion
- ligation
80.4% of human genome linked to
biochemical functions–> bound by a transcription factor
- transcribed
- modified histone
syndrome
bunch of clinical features that are observed in, characteristic of a single condition
Rett Syndrome
- neurodevelopemtn disorder
- mostly females affected
- normal growth and development followed by a slowing down of development
- main effect= loss of motor and intellectual abilities
- microcephaly, seizures, stereotypical hand movements
caused by mutations in the MeCP2 protein
CHARGE Syndrome
colobomas of eyes heart defects atresia of choanae retardation of growth/developemnnt genital hypoplasia geneital hypoplasia ears
CHD7 protein mutations
Rubinstein-Taybi Syndrome
broad thumbs and toes short facial features low set ears ocular abnormaliteis cardiac abnomalities ID carying
CREBPP/P300- Histone acetyltransferase
intellectual disability
big feature of diseases linked to mutations in chromatin proteins
- brain functions depends on the interactive actions of many genes
- sensitive to mass gene expression as well
