MCP 4

Question 0

how do we create a probe for FISH?

Answer 0

DNA/ gene location is chosen and a unique section of this DNA is isolated - one strand of the fragment is labeled with fluorescent dye
-> then we test to make sure it works (hybridized in a metaphase isolation)

Question 1

Fluorescence in situ hybridization (FISH)

Answer 1

combines the best of cytogenetics and molecular diagnosis -> to get results that neither technique can get on their own - instead of dyes- molecular probes are used
-common goal is determine if a gene or specific mutation or chromosomal rearrangement is present or absent using the specific molecular probe

Question 2

What cell phase(s) are FISH performed?

Answer 2

metaphase or interphase

cell preparation just like karyotype prep

Question 3

what are the three basic types of FISH

Answer 3

1. repeat sequences
2. single copy DNA *subtelomere FISH (super specific)
3. Chromosome painting (multi-colored)

Question 4

repeat sequence probe

Answer 4

Usually isolated from telomere or centromere regions- centromere probes are usually used in chromosome enumeration (to detect the gain or loss of specific chromosomes) - true telomere probe ids the six base repeat present at the ends of all chromosomes and will confirm the presence or absence of the telomeric region

Question 5

Single copy or unique sequence

Answer 5

usually isolated from cloned DNA of a disease causing gene or a fragment of DNA known location associated with a particular gene
-used to detect the presence or absence of a gene, gene region or chromosomal rearrangement of interest

Question 6

Subtelomere FISH probes

Answer 6

DNA sequences from the distal ends of chromosomes in regions proximal to the actual telomere regions -> telomere sequences cannot be used bc they are repeats that are the same for all chromosomes
-short arms = green
-long arms = red
allows for the detection of very small cryptic deletions
-3-5% of mental retardation is due to cryptic terminal deletions

Question 7

Chromosomal painting probes

Answer 7

whole chromosome paint are usually a cocktail of many unique DNA fragments from along the entire length of a chromosome such that following hybridization the entire chromosome fluoresces
-most useful in diagnosing complex rearrangements or marker chromosomes

Question 8

Multicolor FISH

Answer 8

type of chromosome painting that is used to detect multiple chromosomes with one hybridization - done with special probes using a fluorescence microscope and computer with specialized software - typical fluorescent microscope you can only see max of 3 colors
with technology we can recognize the 24 diff chromosomes with computer assisted color schemed->
great for detecting translocations, large duplications, or deletions
-cannot identify inversions, small deletions or duplications

Question 9

why do probes have a limited specificity ?

Answer 9

because the probe is only formulated for a very specific part of the effected DNA- the most commonly critically affected to area- so it may fail to detect other absnomalities in the close surrounding areas bc its limited size

Question 10

What FISH when?

Answer 10

• you cant screen all chromosome or loci
• must maximize your results
• if you think you know the disease- start there (unique sequence)
• if karyotype analysis has given you chromosomes use that information (whole paint or specific chromosomes)
• does clinical info help?- developmental delay may be associated with subtelomeric microdeletion

Question 11

Contiguous gene Syndromes

Answer 11

• regions in the genome with clusters of closely associated genes whose normal functions are generally unrelated
• deletion of that region results in multiple phenotypic anomalies (make it hard to peg) that can be described by a particular syndrome
• size of the deletion and number of genes affected may vary from individual to individual such that there may be subsets of phenotypes that are part of a larger syndrome

Question 12

Examples of contiguous gene syndromes

Answer 12

• WAGR- 11p
• Miller-Dieker/lissencephaly- 17p
• williams syndrome-7q
• Velocardiofacial syndrome - 22q
• 1p - syndrome
• prader willi/angelman syndromes

Question 13

WAGR

Answer 13

• deletion encompassing any combinations of genes
• 1/3 with aniridia get Wilms tumor
• 1/50 with wilms has aniridia

w-wilms tumor
a- aniridia
g- genitourinary
r- retardation  
last two seen with larger deletions

Question 14

Williams syndrome

Answer 14

• deletion of elastin gene on proximal long arm of chromosome 7
• features are consistent with elastin gene loss- coarse skin, hair, lack of flexibility in the aorta, supravalvular aortic stenosis -also renal anomolies and skeletal and joint limitations
• they have developmental problems and cannot live independently - may live in group homes-> usually low IQ, musically gifted suck at math, outgoing and friendly
• blue sclera, stellate iris

Question 15

Velocardiofacial syndrome

Answer 15

-second most common syndrome in humans
- 3 Mb deletions which is considered cytogenic micro-deletion on the 22 chromosome
-cleft lip/palate, hypotonia, short stature, learning disabilities, facial anomalies, cardiac anomalies, feeding difficulty at birth, weak immune
system
- 15 % of the time a parent carries the same deletion but may bot be clinically abnormal -> because the 22 chromosome from the other parent may not be able to compensate for the loss like it may in the parent

-

Question 16

Microarray

Answer 16

common types gene arrays ->looking at the DNA sequences of interest, or expression arrays-> gene products to understand which genes are being expressed in a particular cell at a particular time
copy number variation detection via color
- signal equal amounts -> yellow
- less material present (deletion) -> red
-more material present (duplication) -> green

Question 17

Expression Array

Answer 17

What DNA is actually being expressed -DNA fragments of genome placed on slide, RNA extracted from tissue of interest, cDNA is made and labeled wit fluorochrome and this is hybridized to slide
red-> increased expression
green-> decreased expression
black -> median expression

Question 18

Chromosome microarray

Answer 18

-each DNA fragment is directly associated with its location on a chromosome. Hybridization is done just as in gene array but here the data are plotted in order along the lengths of each chromosome - peaks reveal gain (duplication) and valleys indicate (deletion) of DNA

Question 19

what is the first tier of study for individuals with developmental disabilities or congenital anomalies ?

Answer 19

-chromosomal microarray
=> unexplained developmental delay, intellectual disability, autism spectrum disorders, and multiple congenital anomalies

Question 20

Technology comparison

Answer 20

karyotype- large (>3Mb) numerical and structural abnormalities-genome wide
molecular diagnostics- well defined, specific, very small (1-300 bp) mutations, targeted testing
FISH- well defined specific, medium (10Kb-10Mb) mtations; targeted testing
microarray- generalized genome wide screen for small 1 Kb to large mutations. will not detect balanced rearrangements - (other uses of microarray- prenatal diagnosis, pharmocogenetics, mitochondrial disease and personalized medicine)