Genetic Tools Lecture Oct 4

Question 1

What is a restriction fragment length polymorphism?

Answer 1

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

Question 2

WHy can we use restriction enzymes to identify hypermethylation modification of the genome?

Answer 2

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

Question 3

What can we use variable number tandem repeats for?

Answer 3

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

Question 4

What is the cutoff population frequency that makes a SNP a true SNP?

Answer 4

It depends ont he study you look at - some use 1%, others used 5%

Question 5

How can SNPs be used as biomarkers?

Answer 5

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

Question 6

What is a haplotype and why is it useful?

Answer 6

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

Question 7

The newer the allele is in human history, the ______ the length of the hapotype block.

Answer 7

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.

Question 8

Do you need to know the specific gene you’re looking for if you’re following halotypes?

Answer 8

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

Question 9

What causes Prader-Willi Syndrom?

Answer 9

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.

Question 10

What causes Angelman’s Syndrome?

Answer 10

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.

Question 11

What is clinical cytogenetics?

Answer 11

definition: study of chromosomes, their structure and their inheritance, as applied to medical genetics

Question 12

How does one examine a germline karyotype?

Answer 12

create a metaphase spread from T cells grown in culture

Question 13

What are the 4 categories of centromere positions?

Answer 13

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

Question 14

What are the 5 ways you can identify chromosomes within a karyotype?

Answer 14

banding pattern

centromeric position

size

morphology

chromosomes markers

Question 15

Within a karyote, are chromosomes arrange from smallest to biggest or biggest to smallest?

Answer 15

biggest to smallest

chromosome 1 is the biggest chromosome

chromosome 22 is the smallest

Question 16

What does SKY analyses (spectrial karyotyping) utilize?

Answer 16

combinations of FISH probes - this doensn’t require metaphase spreads, and can be conducted on tissue sections

Question 17

What are the two major categories of chromosomal defects?

Answer 17

1. abnormal chromosome number (these are easiest to detect obviously)
2. Defect in chromosomal structure (breakage, translocations, deletions, duplications - not always easy to detect)

Question 18

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?

Answer 18

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)

Question 19

What are some examples of unbalanced chromosome changes?

Answer 19

1. Duplication - can cause partical trisomy; usually from uneven crossing over during meiosis
2. Deletion - can cause partial monosmy; often from uneven crossing over during meiosis - small or large

Question 20

What is the classic example of a cytogenic translocation disrupting a tumor suppressor/activating an oncogene?

Answer 20

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

Question 21

What causes Down Syndrome?

WHat are the symptoms likely related to?

WHat’s the prevalence?

Answer 21

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

Question 22

More specifically, how miRNA possibly related to the underlying molecular defect in Down Syndrome?

Answer 22

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

Question 23

What are some sex-linked aneuploidy disorders?

Answer 23

Trisomy X: XXX in females

Klinefelter Syndrome: XXY in males

Turner Syndrome: X in females

XYY in males

Question 24

What are the three invasive prenatal techniques?

Answer 24

amniocentesis

cordocentesis

chorionic villus sampling

Question 25

How does amniocentesis work?

Answer 25

amniocentesis: removal of amniotic fluid transabdominally by syringe; fetal cells are cultured for diagnostic tests

Question 26

How does cordocentesis work?

Answer 26

cordocentesis: removal of fetal blood from the umbilical cord, more often used when other methods have failed or are ambiguous

Question 27

How does chorionic villus sampling work?

Answer 27

chorionic villus sampling: biopsy of tissue from the villous area of the chorion, can occur 4-5 weeks before amniocentesis

Question 28

WHat are the 4 non-invasive prenatal techniques?

Answer 28

matermal serum screening

US

MRI

Radiography

Question 29

What can we screen for in the first trimester using maternal serum screening techniques?

Answer 29

1. Beta-human chorionic gonadotropin is INCREASED in Down Syndrome
2. Pregnancy Associated Plamsa Protein A is DECREASED in Down Syndrome

Question 30

What can we screen for in the second trimester using maternal serum screening techniques?

Answer 30

1. Alpha-fetoprotein levels. These are decreased in Down’s and increased in neurl tube defects
2. Unconjugated estriol - decreased in Down’s
3. Inhibin - Increased in Down’s
4. Human Chorionic gonadotropin - Increased in Down’s

Question 31

What is ultrasound used to determine prenatally?

Answer 31

fetal age

sex

fetal gross abnormalities

viability

Question 32

In terms of prenatal Down Syndrome diagnosis, what can be identified on ultrasound?

Answer 32

Nuchal translucency (the thickness of neck skin) is increased and can be detected at 10-14 weeks gestation.

Question 33

What are some ways to test for carrier status?

Answer 33

1. 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)
2. DNA analysis - linkage analysis or direct mutaitonal analysis

Question 34

What is the most common autosomal recessive disease in caucasians?

Answer 34

cystic fibrois - 1 in 25 are carriers

Question 35

What is the most common mutation leading to CF?

Answer 35

A 3 base-pair deletion causing the abscence of a phenylalanine at position 508

Question 36

What are the 5 criteria before newborn screening will take place for any given disorder?

Answer 36

1. would treatment be available if the defect is detected?
2. Is there evidence that early detection is of treatment value?
3. Is physical exam not able to detect the defect?
4. Is the test reliable?
5. Is screening cost effective?

Question 37

How is preimplantation screening done?

Answer 37

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

Question 38

Ultimately, what were the two actual culprits for Prader Willi Syndrome and Angelamans’s SYndrome?

Answer 38

PWS: SNORD116

AS: UBE3A

THese are both long noncoding RNAs that regulate other gene expression

Question 39

What are the risks for having Down SYndrome babies?

Answer 39

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!