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Genetic Mutations BTCI
Genetic Mutations BTCI
The following slides explain the molecular biology behind genetics cases included with the Case It v5.03 download. Online descriptions of these cases can be found at http://caseit.uwrf.edu ('access cases' link)
Question #1
REVIEW: What kind of mutation results from a change in a single DNA base? - a base substitution mutation What is an example of a disease caused by this type of mutation? - sickle-cell anemia
Question #2
REVIEW: What restriction enzyme site is eliminated by the sickle-cell mutation? - MstII - cctNagg is the recognition site (N can be any base) - abnormally long fragment results What techniques are used to detect the sickle-cell mutation? - RFLP with restriction enzyme MstII - (RFLP = Restriction Fragment Length Polymorphism) - Southern blotting with sickle-cell probe
Question #3
A mutation that causes the removal of base pairs from a DNA sequence is called what? - a deletion mutation
Question #3 continued
Give an example of a human genetic condition caused by a deletion mutation. - cystic fibrosis is caused by deletion of 3 DNA bases - missing codon in mRNA - amino acid phenylalenine therefore missing from transmembrane protein in lungs
Question #4
What technique is used to detect the cystic fibrosis mutation in Case A of the Case It! exercise? - RFLP in region linked to mutated gene - loss of MspI site (ccgg) - PCR used to amplify linked region of DNA, then cut with MspI - Southern blotting not necessary since not dealing with lots of fragments
Question #4 continued
What technique is used to detect the cystic fibrosis mutation in Case B of the Case It! exercise? - Southern blot with probe specific for mutation - no RFLP necessary (no restriction enzyme needed) - to determine genotypes, must run separately with normal probe and mutant probe, then compare blots
Question #5
A mutation caused by adding base pairs to a DNA molecule is called what? - an insertion mutation
Question #5 continued
Give an example of a human genetic condition caused by an insertion mutation. - Huntingtons disease (chorea) is caused by the repeated insertion of the triplet CAG ...CAGCAGCAGCAGCAG... - more than 50 repeats of the triplet causes disease - progressive degeneration of nervous system
Background Huntingtons
Huntingtons chorea is a neurodegenerative disease characterized by motor, cognitive, and emotional symptoms. The age of onset for symptoms is generally 30-50 years. The genetic basis of the disease is an amplification in a gene with an (as yet) unknown function. A triplet (CAG) is repeated 20-50 times in asymptomatic individuals; having more than 50 repeats is associated with disease symptoms.
Background Huntingtons
This amplification can be detected by restriction enzyme digestion and Southern blot analysis, since the size of the fragment bound by the probe is increased as a result of the amplification of the triplet repeat. Huntingtons disease is considered a dominant disorder, since one copy of the amplified gene appears to be sufficient to cause disease symptoms.
Question #6
What techniques are used to detect mutation for Huntingtons disease? - RFLP with EcoRI restriction enzyme - Southern blot with Huntingtons probe - mutated fragments are larger because of repeats
Question #7
What disease is caused by the deletion of one or more exons in a particular gene? - Duchennes muscular dystrophy (DMD) - characterized by progressive muscle weakness - a sex-linked characteristic - affects primarily males
Question #7 continued
What are exons? - the parts of the DNA sequence that are actually used when a particular protein is made What are introns? - the parts of the DNA sequence not used (ignored) -the introns are cut out of the mRNA, then exons are spliced together to make functional mRNA
Background - DMD
One form of inherited muscular dystrophy, Duchennes, is X-linked and therefore affects primarily males. The symptoms of Duchenne's muscular dystrophy (DMD) include progressive and severe skeletal muscle weakness. A common mutation associated with DMD is a deletion of one or more exons in the dystrophin gene. These deletions can be detected by restriction enzyme digestion and Southern blotting using a combination of probes that will bind to multiple dystrophin exons.
Question #8
What technique is used to detect the DMD mutation? - RFLP with enzyme HindIII to separate exons - Southern blot with DMD probe cocktail - look for missing exons
Question #9
What are two mutations associated with Alzheimers disease? - mutation in codon 693: glutamic acid changed to glycine - loss of a recognition site for restriction enzyme MboII (gaaga)
Background Alzheimer
Alzheimer disease is by far the most common cause of dementia in aging persons. The disease symptoms are identical to other forms of senile dementia, and diagnosis had been possible only at autopsy by the detection of protein clusters called amyloid plaques in the cerebrum. The disease is multifactorial and inheritance patterns are complex. Some forms of familial Alzheimer disease appear to be inherited as autosomal dominant traits, while others are recessive. Spontaneous Alzheimer disease also can occur in the absence of inherited factors.
Background Alzheimer
Mutations in at least four genes have been linked to Alzheimer disease. One of these is the amyloid precursor protein (APP) gene, which encodes the bamyloid peptide found in the cerebral plaques of Alzheimer patients. The function of APP is not yet known, but certain APP point mutations are associated with inheritance of late-onset Alzheimer disease in some families.
Two examples which can be detected by RFLP analysis are the codon 693 Glutamic acid to Glycine mutation and the codon 717 Valine to Isoleucine mutation. The 693 mutation results in the loss of a MboII site, while the 717 mutation results in the gain of a BclI site.
Question #9
- mutation in codon 717: valine changed to isoleucine - gain of a recognition site for the restriction enzyme BclI (tgatca) What technique is used to detect these mutations? - RFLP with MboII and BclI - Southern blotting with APP probe -look for abnormally large fragment (693 mutation) or abnormally small fragment (717 mutation)
Question #10
What are some mutations associated with breast cancer? -AG deletion (185delAG mutation) - TCAA deletion (4184delTCAA mutation) - C insertion (5382insC mutation) Technique used to detect mutations: - DNA amplifed by PCR (already done in Case It! example) - Southern blot using probe specific for mutation (no RFLP necessary) - if mutation present, 80% risk of breast cancer
These mutations can be identified by amplifying portions of the BRCA1 gene by PCR and then using RFLP analysis, direct sequencing, or hybridization with specific probes to detect the presence of mutations.
Question #11
What mutation is associated with phenylketonuria (PKU)? - C to T base-substitution mutation results in wrong amino acid (similar to sickle-cell mutation) - cant metabolize amino acid phenylalanine - phenylalanine present in aspartame (artificial sweetener found in diet soft drinks, etc) - mental retardation and other problems
Background PKU
PKU is a genetic metabolic disease caused by a mutation in the phenylalanine hydroxylase enzyme. In the most common form of PKU, a C to T point mutation causes an arginine to be replaced by tryptophan at amino acid position 408, resulting in an inactive enzyme and incomplete metabolism of phenylalanine-containing compounds such as proteins. The resulting buildup of phenylalanine can cause mental retardation, eczema, loss of skin pigmentation, and other disorders. If detected early, the disease is treatable by excluding foods high in phenylalanine form the diet.
Background PKU
To analyze these cases, use PCR with the PKU primers to amplify a portion of the phenylalanine hydroxylase gene from blood DNA samples. Then use the dot blot procedure (a modification of a Southern blot) to look for the presence of the mutation.
Question #12
What is Fragile X syndrome? - Leading cause of inherited mental retardation
What mutation is associated with Fragile X? - Repetition of a CGG triplet in a gene on the long arm of the X chromosome - Normal genes can have 6 50 repeats - Premutation: 50 200 repeats (no symptoms) - Full mutation: over 200 repeats - Premutations may turn into full mutations
Background Fragile X
Fragile X syndrome is the leading cause of inherited mental retardation. The mutated gene that causes the disorder is called fmr1 and is located on the long arm of the Xchromosome. It is currently unclear whether this trait is dominant or recessive, because both types of expression have been demonstrated.
Background Fragile X
The mutation involves exaggerated repetition of the CGG triplet in a portion of the fmr1 gene near the 5' end. Those with a functional gene have 6 to 50 CGG repeats, whereas those with a full mutation have 200 or more such repeats. Between 50 and 200 repeats of the codon constitute a premutation. An individual with a premutation is considered a carrier, but does not display any symptoms of fragile X. A premutation may undergo additional repetition to generate a full mutation.