Diagnostic Technologies

Question 1

FISH

Answer 1

• 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

Question 2

creating a probe

Answer 2

• 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

Question 3

3 basic types of FISH

Answer 3

• repeat sequence
• single copy DNA-subtelomere
• chromosome painting- multi color

Question 4

repeat sequence probes

Answer 4

• 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

Question 5

single copy probe

Answer 5

• 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

Question 6

subtelomere FISH

Answer 6

• 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

Question 7

chromosome painting

Answer 7

• whole chromosome paints
• cocktail of many unique DNA fragments from along the entire length of a chromosome
• 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
• 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

Question 9

case 1

Answer 9

• 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

Question 10

FISH caviats

Answer 10

• 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

Question 11

what FISH when?

Answer 11

• 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?

Question 12

contiguous gene syndromes

Answer 12

• 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

Question 13

WAGR

Answer 13

• 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

Question 14

williams syndrome

Answer 14

• 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

Question 15

VCFS

Answer 15

• 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

Question 16

VCFS 2

Answer 16

• 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

Question 17

microarray

Answer 17

• 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

Question 18

gene array

Answer 18

• polymorphisms
• mutations
• copy number variation
• won’t detect balanced rearrangements, because amt of DNA is same

Question 19

expression arrays

Answer 19

• 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

Question 20

copy number variants

Answer 20

• 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

Question 21

chromosome microarray

Answer 21

• for deletion of 18, can see excess red- more control DNA annd low green
• for duplication of 5, see excess green-more test DNA

Question 22

case 2

Answer 22

• 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

Question 23

microarray 2

Answer 23

-first tier study in cases of unexplained developmental delay, intellectual disability, autism spectrum disorder, and multiple congenital anomalies

Question 24

tech comparison

Answer 24

• 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

Question 25

other options from microarray

Answer 25

- prenatal diagnosis - pharmacogenetics - mitochondrial disease id - personalized medicine

Question 26

conclusions

Answer 26

- 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

Question 27

next gen sequencing

Answer 27

-specific mutations, sequence variation -gene/chromosome rearrangement -expression profiles VERY complex results