Genetic Tools Lecture Oct 4 Flashcards
What is a restriction fragment length polymorphism?
It an allelic variant that abolishes OR generates a restriction endonuclease recognition site
or it changes the size of an restrictrion fragment length through either an insertion or a deletion
they can be used to distinguish between 2 chromosomes - usually just as a biomarker on southern blot or PCR
WHy can we use restriction enzymes to identify hypermethylation modification of the genome?
Many restriction enzyme sites are mthylation sensitive, meaning they will be turned off by methylation.
This allows us to tell if there has been hypermethylation (and thus silencing) within the promote region of a specific gene of interest
What can we use variable number tandem repeats for?
VNTRs (also called simple sequence length polymorphisms) are streteches of sequence that are just long repeats (like CACACAC).
they make up a significant part of the genomes, but since they’re not part of genes there is no push for them to be conserved.
thus, they are oftem polymorphic in size between chromosomes and inidviduals and can therefore be used as biomarkers for the gene of interest
usually analyzed by PCR
What is the cutoff population frequency that makes a SNP a true SNP?
It depends ont he study you look at - some use 1%, others used 5%
How can SNPs be used as biomarkers?
They are important biomarkers in gene mapping and phamacogenetics
the assumption is that we’ll use SNP chips to detect thousands of SNPs at once, which will detect susceptibility fo a wide range of diseases
What is a haplotype and why is it useful?
Hapotype is a halpoid genotype
this is a large block of SNPs that are cosegregating in the human population (meaning they’re inherited as blocks)
We don’t reproduce fast enough for the genome to be completely mixed up yet, so when we inherit our parents DNA, we inherit large chunks that were the same in our ancestors waaaaaay back when
because we’ve constructed hapotype maps from every major human population, we can associated phenotypeic traits with the presence of specific haplotypes
This is useful in tracing ancestry
The newer the allele is in human history, the ______ the length of the hapotype block.
The larger the haplotype block
the longer an allele has been selected for, the more generations there have been to mix up the nearby DNA sequences.
Do you need to know the specific gene you’re looking for if you’re following halotypes?
No
if a mutation arises in an individual with a distinctive haplotype, you don’t need to know the identify of the gene, you just need to be able to follow the haplotype
What causes Prader-Willi Syndrom?
The gene responsible for Prader Willi SYndrom lies on chromosome 15, and is MATERNALLY IMPRINTED, which means it’s expressed on the paternal chromosome.
The diseases arises when you have a deletion of the gene.
However, it will only occur if the deletion occurs on the PATERNALLY INHERITED chromosome.
The maternally inherited chromosomes is imprinted, so it wouldn’t matter if there was a deltion anyway.
What causes Angelman’s Syndrome?
In this case, the gene is again on Chromosome 15, but this gene is PATERNALLY IMPRINTED, meaning it’s expressed only on the maternally inherited chromosome.
The disease arises if you have a deltion of the gene
It will only cause disease, however, if the deletion occurs on the MATERNALLY INHERITED CHROMOSOME
the gene on the paternally inherited chromosome is imprinted, therefore it doesn’t matter if there’s a deletion on it.
What is clinical cytogenetics?
definition: study of chromosomes, their structure and their inheritance, as applied to medical genetics
How does one examine a germline karyotype?
create a metaphase spread from T cells grown in culture
What are the 4 categories of centromere positions?
metacentric - central centromere and equal size arms
submetacentric - off-center centromere with different sized arms
acrocentric - centromere near one end
telocentric - single arm - only in mice
What are the 5 ways you can identify chromosomes within a karyotype?
banding pattern
centromeric position
size
morphology
chromosomes markers
Within a karyote, are chromosomes arrange from smallest to biggest or biggest to smallest?
biggest to smallest
chromosome 1 is the biggest chromosome
chromosome 22 is the smallest
What does SKY analyses (spectrial karyotyping) utilize?
combinations of FISH probes - this doensn’t require metaphase spreads, and can be conducted on tissue sections
What are the two major categories of chromosomal defects?
- abnormal chromosome number (these are easiest to detect obviously)
- Defect in chromosomal structure (breakage, translocations, deletions, duplications - not always easy to detect)
What is the difference between a balanced and an unbalanced change in chromosome structure?
Which one is more likely to cause a clinical phenotype and why?
Balanced - results in the same amount of genetic material (this may or may not result in a clinical phenotype depending on lwhether it activated or inactivated a gene)
Unbalanced - different amount of genetic material (this usually leads to a clinical phenotype due to inappropriate gene dosage)
What are some examples of unbalanced chromosome changes?
- Duplication - can cause partical trisomy; usually from uneven crossing over during meiosis
- Deletion - can cause partial monosmy; often from uneven crossing over during meiosis - small or large
What is the classic example of a cytogenic translocation disrupting a tumor suppressor/activating an oncogene?
The BCR-ABL chromosome in chromic myeloenous leukemia
there is a translocation of part of chromosome 22 onto chromosome 9
you can see this with FISH
What causes Down Syndrome?
WHat are the symptoms likely related to?
WHat’s the prevalence?
It’s usually cuased by a trisomy 21 which likely results from meiotic non-dysjunction in meiosis 1
it’s letal to 75% of fetuses
it’s the most common chromosomal birth defect with 1/800 births (1/15 in women over 45)
the symptoms are likely caused yb increased gene dosage, but the gene hasn’t been identified - there is some suggestion that a miRNA is responsible
More specifically, how miRNA possibly related to the underlying molecular defect in Down Syndrome?
THere are 5 mRNAs on chromosome 21 what are overexpressed in Down Syndrome patients
miR155 and miR802 downregulated expression of a methyl binding protein called MECP2 on a different chromosome, so petiants with DS have low MECP2
MECP2 is responsible for chromatin gene silencing, so with low MECP2, you get overepxression of CREB1 and MEF2C in Down SYndrome
These are implicated in the CNS and other symptoms in DS
What are some sex-linked aneuploidy disorders?
Trisomy X: XXX in females
Klinefelter Syndrome: XXY in males
Turner Syndrome: X in females
XYY in males
What are the three invasive prenatal techniques?
amniocentesis
cordocentesis
chorionic villus sampling
How does amniocentesis work?
amniocentesis: removal of amniotic fluid transabdominally by syringe; fetal cells are cultured for diagnostic tests
How does cordocentesis work?
cordocentesis: removal of fetal blood from the umbilical cord, more often used when other methods have failed or are ambiguous
How does chorionic villus sampling work?
chorionic villus sampling: biopsy of tissue from the villous area of the chorion, can occur 4-5 weeks before amniocentesis
WHat are the 4 non-invasive prenatal techniques?
matermal serum screening
US
MRI
Radiography
What can we screen for in the first trimester using maternal serum screening techniques?
- Beta-human chorionic gonadotropin is INCREASED in Down Syndrome
- Pregnancy Associated Plamsa Protein A is DECREASED in Down Syndrome
What can we screen for in the second trimester using maternal serum screening techniques?
- Alpha-fetoprotein levels. These are decreased in Down’s and increased in neurl tube defects
- Unconjugated estriol - decreased in Down’s
- Inhibin - Increased in Down’s
- Human Chorionic gonadotropin - Increased in Down’s
What is ultrasound used to determine prenatally?
fetal age
sex
fetal gross abnormalities
viability
In terms of prenatal Down Syndrome diagnosis, what can be identified on ultrasound?
Nuchal translucency (the thickness of neck skin) is increased and can be detected at 10-14 weeks gestation.
What are some ways to test for carrier status?
- Look for biochemical abnormalities in the heterozygous carrier - for example, look for hydrolase enzyme activity to diagnose a Tay Sach’s carrier
(doesn’t always work if the carrier enzyme levels overlap with normal levels) - DNA analysis - linkage analysis or direct mutaitonal analysis
What is the most common autosomal recessive disease in caucasians?
cystic fibrois - 1 in 25 are carriers
What is the most common mutation leading to CF?
A 3 base-pair deletion causing the abscence of a phenylalanine at position 508
What are the 5 criteria before newborn screening will take place for any given disorder?
- would treatment be available if the defect is detected?
- Is there evidence that early detection is of treatment value?
- Is physical exam not able to detect the defect?
- Is the test reliable?
- Is screening cost effective?
How is preimplantation screening done?
For couples using IVF, one blastomere can be removed from the developing embryo to undergo genetic diagnosis.
then they take whatever embryos are unaffected and implant them in the uterus or fallopian tubes
Ultimately, what were the two actual culprits for Prader Willi Syndrome and Angelamans’s SYndrome?
PWS: SNORD116
AS: UBE3A
THese are both long noncoding RNAs that regulate other gene expression
What are the risks for having Down SYndrome babies?
1/800 in live births overall
rate increases to 1/15 in women over 45
If you already have a down child, the risk increases 8-fold
So for a women over 45 who has had a down child already, 1/15 x 8 = 8/15 = about 50% YIKES!
