Genetic Diagnosis Flashcards
Give an overview of the general strategy needed to diagnose genetic disorders
In this application, the size of the repeat region itself is diagnostic: more repeats means larger shade region
- Can measure by gel electrophoresis (after PCR)
- Can measure by column chromatography (after PCR)
- Can measure by Southern blot, cut on either side of the repeat and use a probe to reveal the repeat area
Important:
- Each triplet repeat disorder must therefore have its own diagnostic test
- The repeat change may not be measured (or detected) by ASO, or NGS, or microarray
How does PCR help diagnose disorders?
May be used to detect triplet repeat expansion as occurs in an exon to cause Huntington disease (the CAG repeat translates to a string of glutamine amino acids)
Primers flanking the repeat region will create different sized PCR products
20 -30 CAG repeats are normal
Late onset- 50 repeats
Early onset-100 repeats
Column chromatography May be used to separate PCR products to diagnose a triplet repeat expansion
Explain gel electrophoresis of a patient with Huntington disease
- DNA sample from the patient is obtained
- PCR amplification of the locus containing the CAG repeat
- Agarose gel electrophoresis to separate the DNA fragments based on the size of the fragments
- After staining with ethidium bromide DNA will fluoresce under UV light; can see that shorter fragments move faster than the larger fragments. Can diagnose HD presymptomatically
Number of repeats is estimated by running the PCR products next to a ladder of markers of known size in an adjacent lane in the gel
20 -30 CAG repeats are normal
Late onset- 50 repeats
Early onset-100 repeats
Why can Huntington be diagnosed on southern blot?
The HTT gene is on an autosome so each of us has 2 copies. One from each parent . Southern blot May diagnose triplet repeat expansion disorders
Explain cell treatment for preparation of a standard G-banding karyotype analysis or for FOSH
- Isolation of cells from peripheral blood sample
- Culture of cells in the presence of phytohemagglutinin to stimulate cell division
- Arrest of cells at metaphase by treatment with colchicine or colcemid
- Cells are swelled by hypotonic solution, and burst onto a microscopic slide
- Chromosomes are fixed and prepared for staining and microscopic visualization
Summarize g-banding
Geimsa staining (g-banding ) is. The standard karyotype analysis
G-banding is imaged after binding of Geimsa stain to metaphase stage chromosomes
Some regions stain dark, some light
This technique requires highly experienced cryptologists that are able to differentiate between chromosomes and recognize subtle changes from the normal binding pattern.
Some deletions will be detected by karyotype. The deletion must be bug enough to affect the banding pattern
Describe g-banding
G-band karyotype is a form of whole genome analysis
So, G-banding is a genomic technique. However, it is of relatively low resolution
We can’t always answer this question with clearly
Typically, only very large deletions are visible by G-banding. Usually for a deletion to be detected, it must be larger than 5 Mb. This depends on location, and the normal banding pattern.
Describe Fluorescent in situ hybridization (FISH)
Fluorescently labeled probes can be used to hybridize partially denatured chromosome spreads
- Probes are 100-500 kB long so that many fluorescent tags can be packed onto the probe to allow visualization
- Key point: resolution is improved over G-banding
Hybridization: annealing of complementary ssDNA
Explain two general types of FISH Analysis
- Chromosome-specific unique sequence probes OR gene specific probes(we simply call this FISH):
- Hybridizes to a specific locus on a chromosome (only one locus)
- These probes are useful to probe for submicroscopic deletions, translocations and duplications of genes (hypothesis driven)
- this means that you must have an idea about what you are looking for
- Chromosome painting- or SKY (Spectral karyotype)
- Uses whole chromosome specific probes to paint each chromosome a different(unique) fluorescent color
- These probes are useful for characterizing complex chromosomal rearrangements and identifying the chromosomal origin of rearranged genetic material
- Looks at entire genome
Explain 22q11.2 deletion syndrome
(Velocardiofacial syndrome+(VCFS; DiGeorge); euphemistically called: “that syndrome of many names”
Condition characterized by:
-Abnormal development of parathyroid glands, thymus, conotruncal region of the heart, and cranial facial malformation
Sometimes this condition is seen by G-banding, sometimes not - it’s difficult to interpret. Easier to detect with FISH; or a suspicious karyotype May be confirmed by FISH; Also detected by microarray
Patients with 22q11.2 DS syndromes often present with immunodeficiency symptoms….
The most common reason to suspect thus syndrome is a cardiac anomaly
- Especially conotruncal
- Tetralogy of fallot
Many patients also have an immune dysfunction
- Approximately about 77% have recurrent infections
- Due to improper thymus development
Many other symptoms linked to thus disorder: cleft lip, palate, craniofacial
Features have common cause, makfkrmation during fetal development
What are the karyotype/background info in DiGeirge syndrome?
A patient has symptoms consistent with 22q11deletion ( DiGeirge) syndrome
Karyotype suggests a deletion of chromosome region 22q11. In this example, it is difficult to interpret -In this case, karyotype is ambiguous.
Complete karyotype is shown along with deletion in chromosome 22
Chromosomal fluorescence in situ hybridization reveals the deletion of one chromosomal region 22q11 segment:
- yellow marks chromosome 2
- red marks the region that must be present on both chromosomes to avoid having the disorder (this patient has the deletion)
22q11.2 deletion (DiGeorge) syndrome is confirmed in this patient
Advantage: FSH technology allows better resolution compared to G-banding karyotype
Disadvantage: the geneticist must know what to look for in FISH, as FISH doesn’t query the entire genome
Do FISH require cells to be in metaphase?
Don’t require cells to be in metaphase
Rapid FISH using probes for chromosomes 13, 18, 21, X and Y for the most popular aneuploidy conditions and sex
Since cell culture is avoided, the lab has very fast turnaround time on test. Sometimes called “RAPID FISH “
Explain generation of whole chromosome FISH probes for whole chromosome analysis
- Sequences unique to each chromosome are created and pooled into individual tubes
- Probes unique to each chromosome are created by labeling with a different colored fluorescent molecule (or combinations of fluorescent probes)
- These labelled DNA probes are combined (mixed), and hybridized to metaphase stage chromosome spreads
- Labeled chromosomes spreads are analyzed by fluorescence microscopy either manually or using automated computer systems
Why can’t we see deletions or duplications on spectral karyotype/SKY FOSH?
Will be invisible by this technique bd abuse the chromosome is simply “painted” a single color
A highly aberrant karyotype associated with cells from a late stage cancer
Some of these chromosomal abnormalities wouldn’t be detected by standard karyotypic analysis, some might be.
This technique is used mostly in cancer genetics to visualize genomic instability
