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1) Retinoblastoma is a cancer of the eye
Retinoblastoma (RB) is the first cancer to be linked to a specific genetic defect
RB is the first tumor suppressor gene to be cloned
a. RB is a childhood cancer; the average age of incidence is only 18 months
It affects 1/20,000 children; there are 300 new cases per year
95% of new retinoblastomas occur before the age of seven
b. RB has a good prognosis: over 90% of cases can be cured if detected early enough
enucleation (eye removal) coupled with radiation is the only effective treatment
Most large size tumors are treated by enucleation because the amount of radiation
required to kill a tumor that fills the eye is just too much for the eye to withstand
c. There are two types of RB, hereditary and sporadic
The total experience with retinoblastoma has been that 5 to 10% of cases have
been inherited; 20 to 30% have been new germinal mutations; and 60 to 70% have
been sporadic, i.e., somatic mutations.
1a. Hereditary bilateral (both eyes affected) is inherited from heterozygous
1b. Hereditary unilateral (one eye affected) occurs from a new germinal
mutation; some data suggests a greater chance of this occurring in the father’s
rather than the mother’s germ line
2. Sporadic unilateral cancer is 55-65% of all cases
d. RB is associated with the occurrence of secondary cancers, including:
1. Bone tumors: e.g. Osteogenic sarcoma is increased 500-fold in bilateral
retinoblastoma patients
osteosarcomas can be induced by radiation at the site of enucleation
2. Skin cancers: e.g. Cutaneous melanomas
3. Muscle and connective tissue tumors: e.g. soft tissue sarcomas
4. Brain tumors: e.g. Pinealoblastomas
This suggests that the RB gene functions in tissues other than the retina
2) RB is a tumor suppressor gene
a. RB is inherited as an autosomal dominant trait
all cells of an affect individual are heterozygous,
however, only a small fraction of retinal cells develop into a tumor
this suggests that the cancer phenotype is a cell recessive
i.e. that you need to inactivate both copies of the RB gene to develop a tumor
b. This idea was consistent with Knudson’s two hit hypothesis
1. Knudson modeled the etiology of RB by examining cancer frequencies in the
2. He evaluated the number of unilateral and bilateral cases in hereditary and nonhereditary forms of the disease, and the numbers of tumors found per eye.
Bilateral, hereditary cases occurred at an earlier age of onset
they also typically developed more than one tumor
unilateral, sporadic cases were usually unifocal (one tumor)
3. The data best fit a statistical model whereby RB tumors required two mutations
c. In hereditary RB, one mutation is inherited through the germ cells, and one mutation
occurs in somatic cells (e.g. the retina)
In sporadic RB, both mutations occur in somatic cells
This fit the idea of a recessive tumor suppressor gene
i.e. +/- cells are normal, and -/- cells become cancerous
d. Loss of heterozygosity (LOH) is consistent with the Knudson hypothesis and can
explain the origin of retinoblastoma. Mechanisms of LOH in retinoblastomas include:
1. loss the wild type allele through mutation
2. chromosomal mechanisms that produce homozygous RB mutant cells
a. via nondisjunction
b. via mitotic recombination
e. Frequencies of these mechanisms based on 158 informative cases
1. 4% lose the wild type allele through deletion = mutant
2. 36% through mechanisms that retain heterozygosity at the RB1 locus, such as a
second mutation other than a deletion
3. 60% through chromosomal mechanisms leading to homozygosity for the initial
mutation, such as nondisjunction or mitotic recombination.
f. polymorphic DNA markers along chromosome 13 were used to evaluate LOH
3) Cloning the Retinoblastoma gene
a. cytological information
mapped to chromosome 13q14 because deletions were found in RB tumors
the esterase D gene was deleted in same samples; therefore linked to RB
b. linkage information in pedigrees
esterase D genetically linked to RB
c. positional cloning
1. chromosome “walking” from a known gene, esterase D
walking means isolating overlapping segments or clones of DNA from a
particular region
2. isolate cDNAs from fetal retina libraries using the cloned genomic DNA
this tells you where the expressed genes are
d. analysis of cDNA clones
1. the goal is to identify a cDNA that has the predicted expression characteristics
Use northern blot analysis: probe cellular mRNA with your cDNA
2. the predicted expression characteristics are either loss of mRNA expression or
expression of an altered (i.e. different size) mRNA
this is indicative of a mutation in the gene represented by your cDNA
3. note that RB is expressed in more than just retinal cells
this can explain the occurrence of other tumor types
and suggests that RB function is required in many cell types
e. How do you know when you cloned the right gene?
The gold standard is to revert, or complement, a mutant phenotype with your cloned gene
This was done using retroviruses engineered to express RB protein in RB mutant cell lines
1. Suppress the growth of RB mutant cells
2. Change cellular morphology from “fast growing” to quiescent
3. Block tumor formation in nude mice
Heady days: The promise of RB in cancer treatment; see Abramson’s comments
4) How do you study the molecular basis of the disease?
a. forward versus reverse genetics
1. forward genetics: identify mutations that cause particular phenotypes then
clone gene. e.g. isolation of cdc mutants and the retinoblastoma story
2. reverse genetics: clone the gene then make a mutant to cause phenotype
b. reverse genetics in mice is possible
1. ES cells are pluripotent cells that you can grow in culture
2. Homologous recombination is used to knock out a gene in ES cells
3. Mutant ES cells are turned into a mouse by blastocyst injection
4. Standard genetics is used to recover hetero-or homozygous mutant animals
c. mouse mutants make good, but not perfect, models of human diseases like cancer
1. loss of RB function (i.e. homozygous mutant) is lethal to a mouse
2. mice heterozygous for RB get pituitary tumors
these tumors display LOH at the RB locus
d. why do they get pituitary tumors instead of retinoblastoma?
probably because of compensation from other RB gene family members