Diagnostic Technologies Flashcards
1
Q
FISH
A
- fluorescence in situ hybridization
- combines cytogenetics and molecular diagnostics
- molecular probes are hybridized to chromosomes
- used to be silver
- now fluorescent dyes
- used to determine if a gene, specific mutation, or chromosomal rearrangement is present or absent
2
Q
creating a probe
A
- target is chosen and a fragment of DNA is isolated
- DNA that is unique to the region
- one strand labeled with dye
- probe is hybridized to a metaphase cell preparation and it should bind to same location it came from, then probe can be used
- test wither on metaphase or interphase cells
- slides prepared same as karyotype
- DNA is denatures and the labeled probe is allowed to bind
- the rest of the DNA is counter stained
- make sure to have control
- only one signal on chromosome with deletion
- need to know prob- locus/ chromosome specific
3
Q
3 basic types of FISH
A
- repeat sequence
- single copy DNA-subtelomere
- chromosome painting- multi color
4
Q
repeat sequence probes
A
- probes usually isolated from telomere or centromere regions
- centromere probes used in chromosome enumeration
- true telomere probe recognizes the six base repeat present at the ends of all chromosomes and will confirm if the telomere is there
5
Q
single copy probe
A
- probe usually isolated from cloned DNA or a disease causing gene or a fragment of DNA of known location associated with a particular gene
- used to id the presence or absence of gene, gene region, or chromosomal rearrangement of interest
6
Q
subtelomere FISH
A
- DNA sequences from the distal ends of chromosomes in regions proximal to actual telomere region
- can’t use telomere itself- the same
- DNA must be unique to the chromosome
- short arm in green and long arm red
- allows us to id small deletions and rearrangements that cannot be seen by standard karyotype
- 3-5% of unexplained mental retardation is due to cryptic sub terminal deletions
7
Q
chromosome painting
A
- whole chromosome paints
- cocktail of many unique DNA fragments from along the entire length of a chromosome
- complex rearrangements or marker chromosomes
8
Q
multicolor fish
A
- type of chromosome painting that is used to detect multiple chromosomes with one hybridization
- special probes using a fluorescence microscope and computer with specialized software
- the maximal number of useable colors is three
- need computer assistance to see more than 3 colors
- need more filters
- can detect chromosomes rearrangements if computer gives every chromosome a color, but not id inversions, small deletions or small duplications
9
Q
case 1
A
- 9 year old male with developmental delay and dysmorphic features
- possible VCFS
- subtelomere FISH showed no signal on the distal long arm of 18
- deletion resulting in partial monosomy for 18q
10
Q
FISH caviats
A
- doesn’t cover entire deletion, just critical region
- in a 3 MB deletion, probe may only be 10 KB
- deletion may be present tht can’t be detected by the FISH probe designated for that disease
11
Q
what FISH when?
A
- can’t screen all chromosomes or loci
- maximize results
- if you think you know disease, start there
- if karyotype has given you chromosomes- use that info
- does clinical information help?
12
Q
contiguous gene syndromes
A
- 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 that can be described as a particular syndrome
- WAGR- 11p
- Miller-Dieker- 17p
- Williams- 7q
- VCFS- 22q
- 1p syndrome
- prader-willi/angelman
13
Q
WAGR
A
- 11p
- deletions can affect multiple genes
- phenotype depends on which ones
- about 1/3 children diagnosed with aniridia will also develop wilms tumor
- only 1 in 50 wilms patients have aniridia
- mental retrdation and GU defects seen with larger deletions
- wilms tumor, aniridia, GU retardation
14
Q
williams syndrome
A
- deletion on 7q11.23
- elastin gene
- originally thought to be microdeletion, but is a deletion involving several adjacent genes
- coarse skin and hair, lack of flexibility in aorta, supravalvular aortic stenosis
- developmental problems and can’t live on their own
- thickening of skin
- skeletal and joint limitations
- renal anomalies
- low IQ
- excellent musical skills, terrible with math
- outgoing and friendly
- blue sclera
- stellate iris
15
Q
VCFS
A
- 2nd most common syndrome known in humans
- cleft palate and conotruncal heart defects
- diagnosed when kids have trouble feeding, cardiac defects, and characteristic facial dysmorphologies
- learning disabilities, short stature, and conductive hearing loss are noted as the individual ages
- 3 MB- microdeletion
- chromosome 22
- 40 genes and 8 pseudo genes
- there are repeated sequences that flank the gene, during meiosis, the homologous chromosomes should pair evenly
- repeats are similar- wrong ones could pair and deletions and duplications occur when recombinations happen
- most common is largest-3 MB at two most distal repeats
16
Q
VCFS 2
A
- same 3 mb deletion is seen in majority of cases
- phenotype is variable
- 15% of the time, a parent carries the same deletion but may not be clinically abnormal
- combo of alleles is different than what the parent has
- one parents alleles can’t compensate
17
Q
microarray
A
- gene chip technology
- gene arrays
- expression arrays
- a test DNA is compared to a reference DNA that has a known genetic complement
- DNAs are hybridized and the resulting fluorescent signal is identified and recorded
- data collection is done by specialized equipment
- if DNAs are equivalent, signal should be a composite of red and green that equals yellow
- green means excess of reference DNA, deletion in test
- red means excess of test DNA, and duplication of test
18
Q
gene array
A
- polymorphisms
- mutations
- copy number variation
- won’t detect balanced rearrangements, because amt of DNA is same
19
Q
expression arrays
A
- DNA fragments representative of the genome are placed on the slide
- RNA is extracted from tissue of interest, cDNA is make and labeled with color, and added to slide
- high level of expression is detected by red signal, green indicated decreased gene expression
- can look at same gene in different tissues and see what genes are turned on
- can put it all together as tumor fingerprinter, and clinical testing can be established
20
Q
copy number variants
A
- DNA fragment is directly associated with its location on a chromosome
- hybridization done, data plotted in order along the lengths of each chromosomes
- peaks mean gain and valleys mean loss
21
Q
chromosome microarray
A
- for deletion of 18, can see excess red- more control DNA annd low green
- for duplication of 5, see excess green-more test DNA
22
Q
case 2
A
- 14 year old
- microcephaly
- hemiparesis
- menstrual disorder
- X chromosome anomaly?
- then at 16, some developmental delay noticed
- tested for VCFS, subtelomeres
- then at 24- found deletion of 12p
- 8.8 mb
- 8732 markers
- 40 genes- including SOX5- development
- can’t see just on karyotype
23
Q
microarray 2
A
-first tier study in cases of unexplained developmental delay, intellectual disability, autism spectrum disorder, and multiple congenital anomalies
24
Q
tech comparison
A
- karyotype is large, numerical and structural abnormalities, genome wide
- molecular diagnostics is well defined, specific, very small mutations, targeted
- FISH is well defined, specific, medium mutations, targeted
- microarray-generalized genome wide screen for small to large mutations, will not detect balanced rearrangements
25
Q
other options from microarray
A
- prenatal diagnosis
- pharmacogenetics
- mitochondrial disease id
- personalized medicine
26
Q
conclusions
A
- FISH now an established tool in genetics and oncology
- microarray adds a new dimension to testing
- can generate clinically relevant data that can’t be obtained with other tests
- some uncertainty because we don’t yet know what all of the results are telling us
- new findings will be contributed to national databases to expand the general knowledge of the human genome
27
Q
next gen sequencing
A
-specific mutations, sequence variation
-gene/chromosome rearrangement
-expression profiles
VERY complex results
