Different versions of a gene

Can be detected due to base pair differences (RFLP)

A gene for which multiple alleles exist in the population

ex. ABO blood groups, G6PD variants, etc.

- Allele must be present in at least 1% of population to be considered polymorphic

1 = p2 +2pq + q2

p2 = % people with w.t., functional allele

q2 = % people with non-functional allele

2pq = % heterozygotes/carriers

p + q = 1

(sum of frequency of both alleles in population = 1)

% of chromosomes in the population with a specific allele

ex. 64/100 people have genotype MM

32/100 have genotype MN

Allele Frequency of M = [(2 x 64) + 32] / 200  (200 chromosomes in 100 people)

= 160/200 = 0.8

Centromere is at center of chromosome p arm = q arm

Chromosomes: 

p arm < q arm

Chromosomes: 

Centromere is located at tip of chromatid pair

p-arm is basically non-existent (satellite)

Chromosomes: 13, 18. 21

Polyploidy/Euploidy

Triploidy = 3x all chromosomes; usually fatal in 1st trimester

Tetraploidy = 4x all chromosomes; fatal

Aneuploidy

Monosomy = single chromosome - fatal; except Turners Syndrome (single X chromosome)

Trisomy = 3x single chromosome; autosomal or sex-chromosome

- During gametogenesis with reciprocally/Robertsonian translated chromosomes:

1) Homologous regions pair (forms an +)

2) Can segregate horizontally or on a diagonal

3) Alternate Segregation = diagonal assortment --> results in balanced chromosomal material in gametes (complete chromosomes of each)

4) Normal with fertilization

5) Offspring are translocation carriers

Down Syndrome - 47,XX+21 or 47,XY+21

- most common autosomal trisomy

- Risk: AMA --> increased risk of MI non-dysjunction

- Usually two maternal chromosomes - each have different alleles if NDJ was at MI (mothers and fathers)

- Sxs: mental retardation, low IQ, delayed milestones

- Short stature, depressed nasal bridge, epicanthal fold (above eyes), Simian crease (hand)

- CHDs (mortality)

- Alzheimers-like sxs at a young age

Edward Syndrome - 47,XX+18 or 47,XY+18

-Risk: AMA --> increased risk of non-dysjunction

-Sxs: mental retardation

-clenched fist, overlapping fingers, rocker-bottom feet

- CHDs

- low-set ears, micrognathia (small jaw)

- Often stillborn or early mortality

Patau Syndrome - 47,XY+13 or 47,XX+13

- Risk: AMA --> increased non-dysjunction

- Sxs: Mental retardation

- Polydactyly

- Cleft lip/palate

- Microphthalmia

- Microcephaly

- Cardiac anomalies

*least compatible with life of the trisomies*

45,X

- Non-dysjunction during meiosis

- Sxs:   - Short stature

- Webbed neck, swelling of hands, feet, neck

- Primary amenorrhea; gonadal dysgenesis ('streak ovaries' --> no secondary sex characteristics)

- Often mosaic (some 45,X - no Barr body; some 46,XX; some 47,XXX) - indicates mitotic nondysjunction

- 47,XXY (one X is Barr body - inactive)

- Difficult to dx @ birth - emerges in puberty

- Meiotic non-dysjunction

- Sxs: - Infertility (testicular atrophy)

- Gynecomastia

- Female hair distribution

- Results in gametes with 2 copies of different maternal chromosomes

- Most common type in females

In males: produces:

2 (      ) gametes --> Turner's 

2 ( XY ) gametes --> Klinefelters

- Results in gametes with two copies of the same chromosomes

- In males produces:

2 (     ) gametes --> Turner's

1 ( XX ) gamete --> Triple X Syndrome

1 (  YY  ) gamete --> increased aggression

- Cells show more than one type of genetic composition -- mosaicism

- Occurs during embryonic development

Exchange of genetic material between non-homologous chromosomes (different #s)

Effects: 

- Somatic Cells: transformation to cancer --> uncontrolled growth

ex. Philadelphia Chromosome - (t(9;22)) activation of ABL oncogene --> cancer 

- moves ABL from c#9 to near BCR gene --> creates a new TyrKinase that promotes growth 

- Germline Cells: increased SAB

- During gametogenesis with reciprocally/Robertsonian translated chromosomes:

- Unbalanced translocation that occurs in acrocentric chromosomes (13,14,15,21,22)

- Fusion of 1 short (p) arms of each translocated chromosome (satellites - contain t/rRNA genes)

- Fusion of 2 long (q) arms

- Results in loss of 1 chromosome (2 fused satellites --> ex. 45,XX,-14,-21,+t(14,21))

- Gametogenesis: adjacent or alternate segregation can produced balanced translocation carriers or unbalanced/partial trisomy gametes

- Causes 2-5% DS cases

- Deletion of chromosome 5p -->ex. 46,XX del(5p)

- Caused by spontaneous unequal CO

- Sxs:  high pitched cry, mental retardation, speech problems, microcephaly

DiGeorge Syndrome

(Velocardiofacial Syndrome)

- Microdeletion of chromosome 22q

- Most common microdeletion

Sxs:  CHDs, thymic aplasia (-->immunodeficiency), cleft lip/palate, learning disabilities, narrow palpebral fissures, prominent nasal root

- Deletion of maternal chromosome 15

- Paternally-imprinted genes on C15--> complete loss of function

- Deletion of paternal chromosome 15

- Maternally-imprinted genes on C15 --> complete loss of function

- Microdeletion of chromosome 4p

-Sxs: widely-spaced eyes, prominent nose, abnormal iris, CHDs, mental/developmental delays

- Pericentric - involve centromere

- Paracentric - no centromeric involvement

- Usually balanced and spontaneous (no information loss)

- Changes karyotype banding pattern

[image]

- In meiosis:  homologous pairing of inverted segments + RC can result in duplication/deletion of segments and/or centromeres:

--> acentric = no centromere

--> dicentric = two centromeres

- Loss of one arm + duplication of another arm

ex. X isochromosome - long (q) arms join to form 2q structure, (p) arms form 2p

- Some patients have [1 X isochromosome + 1 normal] :: 3 long arms, 1 short arm --> manifests as Turner Syndrome (aka 45,X)

Loss of telomeric genetic material --> fusion of terminal ends

- No gene loss

- Causes high rate of MAB (meiosis errors)

- Increases with radiation exposure (higher rates of breakage)

H-W Shortcuts:

Autosomal Recessive

#1 Sibling of a known affected individual = 2/3 carrier risk

#2 Parent of a child with A.R. disease must be a carrier

#3 Sibling of a known carrier has a 1/2 chance of being a carrier themselves

#4 Affected parent will always have all carrier children

H-W Shortcuts:

X-Linked Recessive Diseases

q = I

(since only affected males are counted - a female with disease is unlikely and won't change allele frequency enough to be significant anyway)

H-W Shortcuts:

Autosomal Dominant Diseases

I = 2pq

q = I/2 (since p = 1)

1.) Large population

2.) Random mating

3.) No change in mutation rate

4.) No gene flow in/out of population

• Genetic drift in small populations
• Founder Effect: new population establishment by a very small number of individuals from a larger population = limited gene pool
• Gene flow due to population migration
• Incest, consanguinity, inbreeding
• Natural selection

'These Carrier Patients Can Stay Healthy'

Tay-Sachs

Cystic Fibrosis

PKU

Canavan Disease

Sickle Cell Anemia

Hemochomotosis

Thalassemia (a/b)

Xeroderma Pigmentosa

Hurler's Sydrome

MSUD

Alkaptonuria

a-1-Antitrypsin Deficiency

Bloom Syndrome

Xeroderma Pigmentosum

Ataxia Telangiectasia

'Lucky Guys Don't Have R/G Colorblindness'

Lesch-Nyhan Syndrome

G6PD Deficiency

Duchenne MD/Becker MD

Hemophilia A/B

R/G Colorblindness

'Family Members All Have the M.O.A.Ns'

Familial Hypercholesterolemia (FHC)

Marfan's Syndrome

Achondroplasia

Huntington's Disease

Myotonic Dystrophy

Osteogenesis Imperfecta

Acute Intermittent Porphyria (AIP)

Neurofibromatosis

Retinoblastoma (need 2 mutations total)

HNPCC

FAP

BRCA1/2

Li-Fraumeni

- Heterozgosity imparts some selective advantage to individuals

ex. Sickle Cell Anemia and B-thalassemia - heterozygous individuals possess F. malaria resistance

- Changes H-W equilibrium - keeps mutant allele in gene pool at relatively high frequency

Ability to survive and reproduce (i.e. to pass allele to offspring)

ex. Tay Sach - fitness = 0; no survival to reproductive age

ex. Turner Syndrome - fitness = 0; infertility

ex. Huntington's Disease - fitness = average; disease doesn't manifest until after reproductive age in most individuals

 - Random changes in p and q during meiosis

- Much more pronounced in small populations (larger population --> less influence)

- Can fix or extinguish an allele over multiple generations

- Allele amplification when a new, small population diverts from main population or a new allele enters a small population

- Can be coupled to social, religious or geographic isolation --> even more pronounced

ex. Albinism in Hopi Indians in AZ

ex. Tay-Sachs, Gaucher's and Canavan Disease in Ashkenazi Jews

ex. Huntington's in Venezuela village

- Mating with an individual who is a second cousin or closer (i.e share a grandparent)

- More likely to share same disease-causing genes - results in higher incidence of recessive diseases in particular

- Multiple genetic and environmental factors involved in disease process (genetic susceptibility + environmenta influence)

- No simple inheritance pattern

- Genetic component is often polygenic 

- Examples: diabetes, cancer, CAD, mental health, neurodegenerative disorders (ex. Schizophrenia)

- Investigation - use association, population, family and twin studies

Inheritance/expression are dictated by many additive genes at different loci (cumulative)

Influenced by modifier genes

No single gene is dominant or recessive to another

Shows normal/bell curve distribution

ex. Height, blood pressure, intelligence - involve many genes + environmental factors

- Risk of having a disease is higher in one sex than the other

- However, if a female has a disease that is usually  male-predisposed, it means there are more 'bad genes' present - her male relatives have an even higher risk

Liability = all genetic + environmental factors that contribute to a disease/disorder

Threshold = point on normal curve above an abnormal phenotype is expressed (beyond threshold = affected)

- Threshold can be higher for one sex than the other (ex. males = higher rates of CAD and pyloric stenosis - lower threshold; females = higher rate of spina bifida - lower threshold)

- Liability curve for relatives of an affected individual is shifted to the right (i.e. more susceptible to develop the disorder - more 'bad genes' present)

- Threshold stays the same

:: higher # of people above threshold = higher familial incidence of disease

- If more than one affected individual in a family, curve shifts even more

- Risk INCREASES with # of affected individuals in a family 

- Risk INCREASES with severity of defect/disease ('more bad genes')

(this is the opposite of Mendelian inheritance, where severity/frequency don't affect recurrence risk)

- When there is sex predilection for a defect, if an individual from the less-affected sex is affected, the risk for their offspring is even higher

- Recurrence risk for 1st-degree relatives (siblings, offspring) = square-root of I

- An estimate of the % of origin of a disease caused by genetic factors (as opposed to environmental)

(:: will never be 100% for multifactorial diseases)

- Provide an indication of importance of genetic factors in a condition

:: greater heritability --> greater genetic component

[image]

- Study of migrant groups with low disease incidence

After Migration:

- If migrant population maintains low incidence in new population with high incidence --> highly heritable disorder (genetic > environmental)

- If migrant population develops new, higher incidence --> environmental factors > genetic

MZ Twins - single zygote :: same genes, same IU environment

DZ Twins - two zygotes :: 50% shared genes, same IU environment

- Compare incidence in MZ and DZ twins to examine genetic v. environmental factors in multifactorial disease

- Compare Concordance vs. Discordance

In twin studies: presence of a disease in both individuals

- If concordance for MZ > DZ --> disease has a genetic component

- If concordance for DZ = MZ --> caused by environmental factors 

- 100% concordance = single-gene disorder

In twin studies: presence of disease in one individual but not the other

- If a factor is discordant in MZ twins - must be due to environmental influence

- If a factor is discordant in DZ twins - must be due to genetics

 Help differentiate between environmental and genetic disease causation

Ex. Adopted children of schizophrenics have a higher % of schizophrenia because of the large genetic component (same genes, different environment)

Occurrence of some alleles more commonly in individuals affected with a disease than in the general population

- Useful for studying multifactorial diseases

- Identify susceptibility loci - not (+) diagnostic factors for a disease but indicators of susceptibility

ex. HLA B27 - seen in 90% of people with ankylosing spondylitis

- However, only 1% of people with HLA B27 develop ankylosing spondylitis :: there must be environmental factors as well

- Used in association studies to assess linkage between an allele/SNP and likelihood of developing a disease

- Not a (+) diagnostic factor for a disease but an indicator of susceptibility

- Compare entire genome of people with a disease to the entire genome of those without it

- Useful in identifying new susceptibility loci

- Quantification of level of association between a risk factor and a disease

- What are the odds of an affected person having a risk factor compared to an unaffected person?

ex. Odds Ratio = 2 - the risk of person with ankylosing spondylitis having HLA B27 is 2x that of an unaffected person

- Odds Ratio > 1 = association between risk factor and disease

--> if you have a disease - what's the chance you'll have a given risk factor?

- Relates the difference in outcomes for those with and without a susceptibility locus

ex. Relative risk = 20 - chances of developing ankylosing spondylitis in a person with HLA B 27 is 20x higher than in a person without HLA B 27

--> if you have a risk factor - what's the chance you will be affected?

Neural Tube Defects

- Multifactorial

Genetic Factors:

MTHFR (methyl-THF reductase)

MTHFD (methy-THF dehydrogenase)

Previous history of child with NTD

Environmental Factors:

Maternal folate ingestion prior to and during preg

Dx: maternal AFP, U/S to visualize development

- Autoimmune destruction of B-cells of pancreas

- Controlled w/ insulin replacement injections

- Large familial/genetic component

- 20 associated susceptibility loci:

ex. HLA-DQ2/DQ8 - chromosome 6

ex. Insulin gene - VNTR polymorphism

- Peripheral insulin resistance; variable degrees of secretory defect

- Adult onset

- Pts usually obese

- Multifactorial with a large environmental component :: tx and prevention usually includes lifestyle changes

- Multiple genetic components, include:

ex. PPARG (peroxismal proliferator-activated receptor-gamma)

ex. Glucokinase (in Type II MODY - maturity-onset diabetes of the young)

- Most common cause of dementia

- Multifactorial disease

- Dx: amyloid deposits of amyloid-B-precursor protein and Tau protein (MTAP) in neurofibrillary tangles and plaques

Two Forms:

1.) Familial - dominant inheritance

- Early-onset

- C1 = Presenilin-2

- C14 = Presenilin-1

- C21 = amyloid-precursor protein

2.) Sporatic

- Late-onset

- Susceptibility locus = ApoE4

- Environmental factors: poor CV health, age, head injury, smoking, aluminum exposure

- Autosomal recessive

Mutations: (1000+ discovered)

- FΔ508 deletion --> loss of Phe

- Causes misfolding and degradation of CFTR protein in mucus epithelium

- R117H single BP mutation - from Arg --> His

- Milder phenotype - CFTR present but with reduced Cl- conductance capacity

Sxs:  - variable severity

- Lung/liver/pancreatic disease,

- Elevated sweat Cl

- Male infertility

- Autosomal Dominant

- Dominant-Negative mutations

- Defect in Type I collagen formation

Mutations:

- Primarily due to de novo mutations (esp in most lethal cases) :: highly variable location/type of mutation (heterogeneity)

OI Type I - usually introduction of premature stop codon in COL1A1 gene --> ↓mRNA stability

OI Types II-IV - glycine substitution in pro-α chain --> prevents normal helical structure

Sxs: - variable severity

- Blue sclera

- Bone fx with minimal trauma

- Dentinogenesis imperfecta (DI)

- Eventual hearing loss

 Dominant mutated/altered gene product is more deleterious than having no gene product at all (null mutations)

ex. OI - defective pro-α collagen

- X-Linked Recessive

- Very low reproductive fitness

Mutation:  very large deletion in Dystrophin gene @ Xp21 - results in defective dystrophin protein

Sxs:

- Progressive muscle weakness - proximal > distal

- (+) Gower's Sign - using hands pushing on knees to stand up

- Respiratory/cardiac muscles --> death from heart/respiratory failure

- Female manifesting heterozygotes with skewed X-inactivation --> weakness

α-1 Antitrypsin Deficiency

(α1ATD)

- Autosomal recessive

Mutations:

- Chromosome 14 - SERPINA1 gene

- Mutant Z allele (PI*Z- most common) - causes α1AT to accumulate intracellularly in hepatocytes (not secreted)

- PI*M, PI*S (normal alleles)

- PI*Mmalton, PI*Siiyama (mutant)

Sxs:

- COPD (early onset w/ smoking)

- Liver disease (usually in homozygous PI*ZZ)

Qualitative decline in hemoglobin

Mutation in nucleotide sequence produces abnormal protein 

Hemoglobin

Fetal v. Adult

Fetal = 80% HbF = α2γ2

= 20% HbA   

Adult = 97% HbA = α2β2

= 2.5 % HbA2

- Shift from γ to β begins @ ~35 wks

Quantitative decline in hemoglobin

Mutation causes decrease/absence of a globin chain

Pathology due to imbalance in α:β ratio

 - Autosomal Recessive

Mutation:

- GAG --> GTG(Glu --> Val) @ position 6 on HbA

- Creates HbS (abnormal protein)

- GAG --> AAG(Glu --> Lys) @ position 6 on HbA

- Creates HbC (milder form, well compensated)

Pathology:

- HbS aggregates inside RBCs - form inflexible rods that distort shape

- RBCs unable to squeeze through capillaries

- Decreased RBC lifespan

Sxs:

- ↓O₂ delivery to tissues

- Chronic anemia, hepatosplenomegaly

- Sickling Crises - severe pain from reduced blood flow

- HbA/HbS (compound heterozygote)

- RBCs retain flexibility (enough HbA to compensate)

- No sickling crises - only pain with anoxia (usually leads to dx)

- Imparts heterozygote advantage for malaria

- Autosomal Recessive

- Mutation in β-globin (HBB) gene on chromosome 11 --> reduced synthesis

- Excess α-chains bind RBC membrane and form toxic aggregates

- Variable phenotypes

- Alleles: 

B = w.t.

Bo = no HbB synthesis

B+ = low level HbB synthesis

3 Forms:

1.) B-Thalassemia Major

2.) B-Thalassemia Intermedia

3.) B-Thalassemia Minor

- Autosomal Recessive

- Mutation of α-globins --> reduced synthesis

- Excess 

- Probably due to unequal CO

2 Forms:

1.) Hb Bart Hydrops Fetalis Syndrome

2.) Hb H Disease

B-Thalassemia Major

(aka Cooley's Anemia)

- Homozygous mutant or compound heterozygous

(Bo/Bo, B+/B+ or B+/Bo)

- No HbB synthesis

Sxs:

- Severe microcytic anemia

- Iron overload

- Splenomegaly

- Marrow expansion

- Growth failure --> Death

Tx:

- Frequent RBC transfusion

- A-Thalassemia

- Most severe - 4/4 α-globins are deleted

Sxs:

- Severe anemia

- Fetal edema, ascites, pericardial/pleural effusion

Dx: U/S @ 22-28 wk (13-14 wk with ↑ NT)

--> neonatal death

- A-Thalassemia

- Deletion of mutation of 3/4 α-globin alleles

Sxs:

- Microcytic hemolytic anemia, hepatosplenomegaly

- Mild bone changes - 'hair-on-end' skull

--> compatible with survival to adulthood

- X-linked recessive

- Deficiency or lack of clotting factors --> abnormal/prolonged hemorrhage

- High frequency of new mutations

Three Types:

1.) Hemophilia A (aka Classic Hemophilia)

2.) Hemophilia B (aka Christmas Disease)

3.) Hemophilia B Ledyen (rare)

- Skewed X-inactivation (Lyonization) can produce Affected Heterozygote females --> v. mild symptoms, if any

- X-Linked Recessive

- Heterogenous mutation in Factor VIII gene - usually an inversion of Intron 22 (can also be insertion, deletion, frameshift, etc.)

Sxs:

- Hemorrhage into joints/muscles --> arthritis, necrosis

- Excessive wound bleeding

- Severity is proportional to Factor VIII levels

Tx:

- Frequent IV plasma Factor VIII supplementation

- Acquired

- Cause cell damage, death or cause cancer

- Mutation is passed to daughter cells only (within same cell line)

- No vertical transmission of mutation to offspring

ex. UV rays --> melanoma of skin cells

ex. Philadelphia translocation of C22,9 --> CML in patient, not transmitted to offspring

- Occurs in germ cells (sperm or ova)

- Transmitted to offspring with fertilization

- Mutation is present in all body cells of offspring

ex. Spermatogenic mutation introducing Osteogenesis Imperfecta --> afflicted offspring

*spermatogenesis introduces many opportunities for mutation*

- Change of one AA can introduce:

1.) Missense Mutations - changes an AA in product

ex. Sickle Cell - Glu-->Val

2. Nonsense Mutations - introduces new stop codon

ex. CAG-->UAG

      (Glu)-->(stop)

3.) Silent Mutations - no effect on gene product

3 base D/I --> protein contains -/+ one AA

- Can be problematic -

- ex. CF = ΔF508 Serine deletion

- this AA is crucial to translocation of CFTR to cell surface :: defective after mutation

2 base D/I --> frameshift; entire downstream structure is changed

1 base D/I --> frameshift; entire downstream structure changed

- Larger deletions can eliminate entire functional regions (ex. MD - large dystrophin deletion)

- Mutations (point/insertion/deletion) can introduce new splice sites in introns

ex. Thalassemia - single BP mutation creates an alternate splice acceptor site on HBB gene --> abnormal/absent HgB

Mutations in:

- Promoter

- 3'-UTR

- 5'-Poly-A tail

Can change expression levels of a gene

Single BP shift from

Purine --> Purine (A<->G)

OR

Pyrimidine --> Pyrimidine (C<->T)

Single BP shift from

Purine --> Pyrimidine

(or vice versa)

ex. A --> T

- Less common than transition mutations

- CG repeat regions involved in methylation

- 'Hotspots' of mutation where C --> T during replication

A single BP change that doesn't change the AA encoded by that codon

ex. CCA --> CCG

     Pro --> Pro

A single BP change that affects what AA is coded for  in the DNA

ex. AGC --> GGC

    Ser --> Gly

- Highly sequence-specific cutting enzyme

:: can be used to detect SNPs at specific target sites

(will no longer cut if BP sequence has been changed   --> generates longer fragments than w.t.)

STR = Short Tandem Repeat

SSR = Simple Sequence Repeat

- 1-6 bp repeated sequences

- Used in gene marking/RC mapping

- Variable # of repeats between individuals

--> used to determine paternity and for DNA identification 

(most useful RP length for DNA fingerprint is 4 BP)

Copy-Number Variant (CNV)- large segment exists multiple times in a genome compared to a reference

Copy-Number Polymorphism - a CNV that occurs in at least 1% of the population

Segmental Duplication - two or more copies of a segment within a genome (can also be CNVs)

Inversion - segment of DNA that is reversed in orientation compared to the rest of the genome

Translocation - change in position of a segment within a chromosome (doesn't change internal sequence of segment)

- VNTRs and STRs

- Transcriptionally inactive

- Involved in regulation of gene expression

Changes that involve segments > 1kB:

- Insertions
- Deletions
- Duplications (Copy-Number Variants, Segmental duplication)
- Inversions (pericentric or paracentric)
- Translocations

- Can interrupt or up-regulate gene expression depending on location/type of structural variation

ex. Insertion downstream from a constitutive promoter --> overexpression

ex. Duplication --> increased expression (multiple sites of transcription)

A set of linked polymorphic genes that are inherited as a unit

- located at different loci on a chromosome

- G1 arrest by p53 (prevents cell cycle progression before DNA repair)

- Cell death if damage is extensive/cannot be repaired

- Aging (normal)

- Cancer

1.) UV Light --> thymidine dimers 

2.) Radiation --> double-stranded breaks, ring chromosomes

3.) Cellular Metabolism (ex. ROS, normal aging effects)

4.) Chemical Exposure (ex. nitrous acid)

5.) Replication Errors

DNA Pol I (prokaryotes) has 3'-5' exonuclease activity 

:: removes mis-paired base immediately after addition and replaces with correct base

DNA repair of 1-5 nucleotide segments:

1.     Recognition of damage

2.     Removal of base

3.     Cleavage of strand at removal site

4.     Gap filling by DNA Polymerase using other strand as template

5.     Re-sealing by DNA Ligase

ex. to remove thymidine dimers caused by UV damage

DNA repair mechanism for 20-30 nucleotide segments:

1.     Recognition of dimer

2.     Removal of 20-30 bases

3.     Gap filling by DNA Polymerase using other strand as template

4.     Re-sealing by DNA Ligase

-  When mismatch occurs in daughter strand --> mismatch repair

system recognizes error

- Can tell parent/daughter strand by methylation status (daughter

strand not yet methylated)

- Corrects error based on parental template 

- Involves MSH2 and MLH1 proteins:

--> Mutations in these proteins causes HNPCC (hereditary non-polyposis colorectal cancer)

Causes:

- Autosomal Recessive

- 100% penetrance

- Defect in nucleotide excision repair (20-30 bp segments)

- Locus heterogeneity (7 genes --> same phenotype)

Sxs:

- Increased sunburn, freckles, sun blisters

- Conjunctivitis

- Increased skin cancer risk

- Some neurological abnl

- Cumulative + irreversible DNA damage

Causes:

- Autosomal Recessive

- 100% penetrance

- Mutation in BLM gene - C15q (DNA helicase) --> results in increase in double-stranded breaks/CO/RC events during mitosis

Sxs:

- Facial rash w/ sunlight exposure

- High incidence of early onset cancer (leukemia, lymphoma, virtually any organ; 50% before age of 20)

Tx:

Prevention of sun exposure

Cancer screening

Ataxia Telangiectasia

(aka Louis-Bar Syndrome)

Causes:

- Autosomal Recessive

- 100% penetrant

- Mutation in ATM gene - C11q 

(ATM usually blocks cell division when DNA damage is present - similar to p53)

Sxs:

- Ataxia

- Telangiectasia (dilated small surface BVs)

- Increased cancer risk (lymphoma, leukemia, melanoma, breast, ovary, stomach)

- Heterozygote females show increased BRCA risk

Tx:

- Radiation = contraindicated

- X-rays only for Dx

- Frequent screening

HNPCC

(hereditary non-polyposis colon cancer)

Causes:

- Autosomal dominant

- 90% penetrant

- Mutations in MSH2 and MLH1 (C2 and C3)

--> decreased mismatch repair = DNA damage

- Results in microsatellite instability

- 2% colon cancer cases

Sxs:

- Early onset (45yo)

- Proximal colon tumors

- Increased risk of extracolonic cancers (endometrial, ovary, stomach, urinary, etc.)

Dx:

- Use Amsterdam Criteria to determine genetic v. sporatic cause

 Used to determine likelihood of genetic causation in cancer (ex. HNPCC)

- Not diagnostic for genetic causation

Qualifications:

1.) 3+ relatives in 2+ generations with verified CRC

2.) At least one case is a 1st degree relative of another

3.) At least one case has onset by age 50

4.) Exclude FAP (familial adenomatous polyposis)

- Unstable short repeat regions

- Caused by failure of DNA repair

- Found in 95% HNPCC tumor cells

- DNA repair gene found on C17q21

- Mutations linked to breast, ovarian cancer

- Autosomal Dominant inheritance

- 500+ mutations known: nonsense, missense, frameshift, splice-site

- High freq. in AJ populations (1/40 carrier for either BRCA1 or 2)

- Female BRCA lifetime risk = 56-85%

- Higher secondary female risk than BRCA2

- Lower male risk than BRCA2 (1-2%)

- Higher OVCA risk than BRCA2 (28-44%)

Cancer

(general characteristics)

- Multi-step process

- Loss of growth inhibition (contact, metabolic, etc.)

- Evasion of apoptosis

- Increased angiogenesis

- Loss of DNA repair function

:: Uncontrolled cell growth/division = tumor

- A normal gene that can become an oncogene due to

mutations or increased expression

- Codes for proteins that help to regulate cell growth and

differentiation – encourage cell division

- Often involved in signal transduction and execution of mitogenic signals, usually through their protein products

- Proteins that suppress mutations in proto-oncogenes that lead to accelerated cell division --> recognize and limit mutated cell division

ex. p53, Rb

- Mutations in tumor-suppressor genes that cause cancer are usually loss-of-function mutations

- Cytosolic signal transduction protein (intracellular signaling)

- GTP-ase

- Mutation can lead to signaling errors --> cell overgrowth

- DNA binding transcription factor that facilitates progression from G1-->S

- Mutation can lead to over-expression - cell cycle progression even w/ DNA damage

- ex. amplified in BRCA

- ex. Burkitt Lymphoma - translocated to IgG gene --> overexpression

- Mutant protein that causes CML

- Created by t(9,22) - (Philadelphia translocation)

- Places ABL protein downstream from promoter of BCR --> fusion protein with tyrosine kinase function (gain-of-function mutation)

-  Inhibited by Imaniteb mesylate

- Oncogene implicated in Follicular Lymphoma

- Anti-apoptosis factor --> mutation leads to overexpression and :: increased inhibition of apoptosis

Ab that binds Her2 receptor on cell surface

:: EGF cannot bind Her2 receptor --> blocks downstream signaling for cell growth/DNA replication

- Used in combination with other drugs to treat BRCA

= Human Epidermal GF-Receptor 2

- Membrane receptor

- EGF binds at surface --> protein signals cell to grow and divide

- Over-expressed at cell surface in BRCA tumor cells :: increased signaling for division (amplification mutation)

- Inhibited by Herceptin

- Caused by Philadelphia translocation (t(9,22))

- Mutation --> mutant fusion protein ABL-BCR with tyrosine kinase function that stimulates cell division

- Dx:  FISH detection of translocation

- Tx: Imaniteb mesylate - blocks TK and stimulates apoptosis in mutant cells

- Translocation mutation t(8,14)

- Relocates c-MYC gene to IgG locus --> overexpression of MYC (TF that promotes cell cycle progression) --> increased cell division

- Mutation in BCL2 proto-oncogene

- Mutation results in over-expression of anti-apoptotic factor --> increased cell division

- Tx for CML

- Binds BCR-ABL tyrosine kinase as ATP-analog

- No phosphorylation of substrates can occur

:: inhibits uncontrolled cell division, promotes apoptosis

- Tumor suppressor gene

- Regulates cell-division and apoptosis by acting as a TF --> suppresses cell cycle promoters and expresses cell cycle inhibitors

-70-80% cancers exhibit p53 mutations

- WT1

- Tumor suppressor gene

- Mutation leads to nephroblastoma (early onset kidney disease)

-Autosomal dominant

- Family of p53 mutations

- Variable expressivity --> affected individuals exhibit high rate of tumor development at a young age (ex. bone, breast, soft tissue, brain cancer, melanoma, etc.)

If 1st mutation is germline (i.e. inherited, present in all cells), 2nd (somatic/acquired) mutation is more likely to cause cancer

(Loss of Heterozygosity)

ex. Rb --> retinoblastoma

Occurs in heterozygous mutant individuals (with germline mutation in one allele)

- Occurrence of a 2nd mutation (somatic) causes loss of heterozygous w.t. status --> now has 2 different mutations

 --> Loss of functional/w.t. allele (via deletion, unbalanced translocation, point mutation, etc.)

- Caused by two mutations in Rb (tumor-suppressor gene) - one in each allele

Mechanism:

1.) Germline mutation in Rb --> inherited in a dominant pattern

2.) Functions as a recessive allele - i.e. remaining functional allele can compensate

3.) Additional acquired/somatic mutation in w.t. Rb gene --> loss of Rb function completely --> disease phenotype

Sxs: early onset deteriorating vision, clouding of eyeballs

FAP

(familial adenomatous polyposis)

- Autosomal dominant

- Mutation in APC gene - C5q (= tumor-suppressor gene)

+ mutations of other oncogenes (progressive)

Sxs:

- 100% lifetime CRC risk (90% by 45yo)

- 1000s of benign polyps

- Increased risk of extracolonic cancer

- CHRPE (congenital hypertrophy of the retinal pigmented epithelium)

- 1^st ‘hit’ in Two-Hit-Hypothesis was 

inherited/germline

- Only takes one more mutation to cause loss of

both genes

- High-risk

Indicators:

1. Young age of onset

2.  Multiple tumors

3.  BL tumors

4.  Autosomal dominant inheritance pattern

- Two somatic mutations have occurred (2-hit hypothesis) to result in cancer

- Much less common

- Compare tumor and normal cell gene expression

- Can reveal:

- Pathogenesis/progression of tumor

- Differentiation genes

- Drugs sensitivity genes

- Risk factors

- Novel targets for therapy

- Small non-coding RNAs

- Regulate translation and degradation of mRNA

- Most cancers show altered miRNA expression/function (ex. epigenetic silencing of tumor-suppressing miRNA)

- Potential sites for disease markers/drug therapy

Examples:

1. Deamination -->↑ rates of mutation
2. UV absorption -->↑ rates of mutation
3. Carcinogens -->↑ rates of mutation
4. Methylation --> gene inactivation

ex. methylation of promoters of repair genes 

--> microsatellite instability, decreased DNA repair