Diagnostic Technologies

Question 1

Fluorescence In Situ Hybridization (FISH)

Answer 1

• combines the best of cytogenetics and molecular diagnostics to obtain information that cannot be obtained by either technology alone
• molecular probes
• used to be tritium then fluorescent dyes
• the most common goal is to determine if a gene, a specific mutation, or a particular chromosomal rearrangement is present or absent, so the molecular probe(s) used must be well characterized and specific to the locus being examined

Question 2

FISH technology

Answer 2

• to create a probe a target like DNA gene or location is chosen and a fragment of DNA is isolated
• this should be DNA that is unique to the region. One strand of the fragment is labeled with a fluorescent dye
• to confirm that the probe works, it will be hybridized to a metaphase cell preparation and it should bind to the same location from which it was derived. Once an appropriate probe is chosen, testing can take place
• FISH can be performed on either metaphase or interphase cells, and slides are prepared just as they would be for a karyotype analysis
• the DNA on the slies is then denatured and the fluorescently labeled singe stranded molecular probe is allowed to hybridize to the chromosomal DNA
• the rest of the DNA is counterstained with another flurochrome to allow visualization of the entire chromosome complement using a fluorescent microscope

Question 3

FISH probes and multiple loci

Answer 3

• the original FISH probes hybridized to a single locus, but this created difficulty in interpretation of the results
• the was never clear if the missing signal was due to a disease related issue, such as chromosome deletion or if the loss was a technical error such as failure of probe to bind to target
• a control probe was localized to a different region of the same chromosome in all the assays as a hybridization control
• usually test and control labeled with different colored fluorochromes
• a person with a deletion should have two signals on one chromosome but only a single signal on the deleted chromosome

Question 4

Parameters of probe

Answer 4

• it only detects the site from which it was derived
• therefore if the probe is specific to chromosome 7 at band 11.23, it will detect abnormalities of that locus but NO others
• thus, a single FISH assay does not provide the same type of genomic information available by karyotype analysis, but it may provide more detailed information about one specific locus

Question 5

3 Types of FISH

Answer 5

• repeat sequences
• single copy DNA- subtelomere FISH
• chromosome painting- multi-color

Question 6

Repeat Sequence Probes

Answer 6

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

Question 7

Single copy probe

Answer 7

• isolated from cloned DNA of a disease-causing gene or a fragment of DNA of known location associated with a particular gene
• this type of probe is used to identify the presence or absence of the gene, gene region, or chromosomal rearrangement of interest

Question 8

Subtelomere TISH

Answer 8

• DNA sequences from the distal ends of the chromosomes in regions proximal to the actual telomere regions
• telomeric sequences themselves cannot be used since these are repeat sequences that are the same of all chromosomes
• the DNA used must be unique to the chromosome and to the specific arm of the chromosome
• the short arm probes are green, red is long arm
• important because it allows us to identify very small (cryptic) deletions and rearrangements that cannot be seen by standard karyotype analysis
• it is known that the subtelomere regions of the chromosomes are gene rich, and that 3-5% of unexplained mental retardation is due to cryptic terminal deletions

Question 9

Chromosome painting

Answer 9

• whole chromosome paints
• cocktail of many unique DNA fragments from along the entire length of a chromosome such that following hybridization, the entire chromosome fluoresces
• this type of probe is more useful in identifying complex rearrangements or marker chromosomes
• if the patient has an abnormal chromosome with extra material origin, it may be possible to use chromosome painting to identify the source of the extra DNA

Question 10

Multicolor RISH

Answer 10

• type of chromosome painting that is used to detect multiple chromosomes with one hybridization
• this is done with special probes using a fluorescence microscope and computer with specialized software
• for a typical fluorescent microscope, the maximum number of useable color is three (one target sequence, one control sequence, and one counter stain)
• combinations of fluorochromes have been developed that allow detection of each of the 24 different chromosome followed by a unique color assignment by the computer. Although this technology is very good at detecting chromosome rearrangements (translocations, large duplications or deletions), it cannot identify inversions, small deletions, or small duplications

Question 11

FISH facts

Answer 11

• probes do not cover the entire deletion- just the critical region
• for example for a 3 MB deletion, the probe may be only 10 KB (velocardiofacial syndrome)
• therefore, a deletion may be present that cannot be detected by the FISH probe designed for that disease

Question 12

What FISH when?

Answer 12

• you can’t screen all chromosomes or loci
• 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 chromosome paint or unique sequence will identify a particular region of a chromosome)
• does clinical info helps?- developmental delay may be associated with a subtelomeric microdeletion

Question 13

Contiguous Gene Syndromes

Answer 13

• 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/Lissencephaly-17p
• Williams syndrome-7q
• Velocardiofacial syndrome-22q
• 1p-syndrome
• Prader Willi/Angelman syndromes

Question 14

WAGR

Answer 14

• one of the best known contiquous gene syndromes
• located on the short arm of chromosome 11
• can affect one or more of the genes aligned in tantem
• Wilms tumor
• aniridia
• genitourinary
• retardation

Question 15

Williams syndrome

Answer 15

• associated with the deletion of the elastin gene on the proximal long arm of chromosome 7
• deletion involving several adjacent genes
• absence of elastin: coarse skin and hair, lack of flexibility in the aorta, supravalvular aortic stenosis
• they have developmental problems and cannot live on their own
• skeletal and joint limitations
• renal anomalies
• usually low IQ
• excellent musical skills but terrible with math
• outgoing and friendly
• blue sclera

Question 16

Velocardiofacial syndrome

Answer 16

• learning disabilities
• hypotonia
• short stature
• cleft lip and/or palate
• facial anomalies
• cardiac anomalies
• feeding difficulty at birth
• weak immune system
• 20% of affected individuals can see deletions on karyotype analysis
• FISH- 2 signals per chromosome 22, but a VCFS patient will have one chromosome with 2 signals and the other with only 1 signal- interstitial microdeletion on 22, 40 genes and 8 pseudogenes
• error due to unequal crossing over
• the same 3 MB deletion is seen in the majority of cases
• phenotype is variable
• 15% of the time, a parent carries the same deletion but may not be clinically abnormal
• different phenotypes in children because their other chromosome 22 will be from other parent and may not be able to compensate

Question 17

Microarray

Answer 17

• gene chip technology
• most common types are gene arrays or expression arrays
• test DNA is compared to a reference DNA that has a known genetic complement
• the DNAs are hybridized and the resulting fluorescent signal is indentified and recorded

Question 18

Data interpretation

Answer 18

• if the DNAs are equivalent, the signal should be a composite of red and green that generally fluoresces as yellow
• a green signal means there is an excess of the reference DNA, thus there must be a deletion in the test DNA
• a red signal means there is an excess of the test DNA, thus there must be a duplication in the test DNA
• most common types of genetic microarrays are 1) DNA (gene) arrays
  2) Expression arrays (RNA)
  3) chromosome arrays

Question 19

Gene arrays

Answer 19

• genes (polymorphisms)
• mutations
• copy number variation
• depending on the DNAs placed on the chip, the analysis can identify genetic polymorphisms, specific mutations, or copy number variation
• this type of assay will usually not detect balanced rearrangements because the total amount of DNA is conserved and no change in the relative amounts will be detected
• if the rearrangement happens to break within one of the gene related polymorphic sequences, this could be detected as a polymorphic variant

Question 20

Expression Assays

Answer 20

• red increased expression
• green decreased expression
• black median expression
• RNA is extracted from a tissue of interest, cDNA is made and labeled with a flurochrome, and this is hybridized on the slide

Question 21

Chromosome Microarray

Answer 21

• it is possible to view the copy number variants in association with the chromosomes
• each DNA fragment is directly associated with its location on a chromosome
• hybridization is done just as in a gene array, but here the data are plotted in order along the lengths of each chromosome
• peaks reveal gain (duplication) and valleys indicate loss (deletion) of DNA

Question 22

14 year old girl microcephaly

Answer 22

• microcephaly, hemiparesis, menstrual disorder- karyotype normal
• age 16- developmental delay in addition, no anomalies
• age 24 microarray- 8.8 MB deletion on short arm of 12
• large- SOX5 gene causing probems

Question 23

Technology Comparison

Answer 23

• Karyotype- Relatively large numerical and structural abnormalities; genome wide
• Molecular Diagnostics: well defined, specific, very small (1-300 bp) mutations; targeted testing
• FISH- well defined, specific, medium mutations; targeted testing
• Microarray: generalized genome wide screen for small to large mutations. Will not detect balanced rearrangements

Question 24

What tests to order

Answer 24

• Known genetic syndrome- Karyotype, FISH
• Clinical features suggestive of a genetic defect without clear association with a known syndrome- Array
• Diseases with known molecular mutation- Molecular
• Known mutations >10 Kb- FISH, Molecular, Array
• balanced rearrangement- karyote, FISH, molecular
• genomics screen looking for cryptic anomalies- Array
• Developmental delay, autism- array
• Mosaicism- FISH, Array
• UPD- Molecular, Array
• Consanguinity/identity by descent- array

Question 25

Other options with microarray

Answer 25

- prenatal diagnosis - pharmacogenetics - mitochondrial disease identification - 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 cannot be obtained with other tests available - 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