Cytogenetics - Techniques of Analysis Flashcards
What techniques are used to sequence the whole genome? (3)
- G banding
- Next generation sequencing
- Microarrays
What are the main targeted tests? (3)
- FISH
- MLPA
- QF-PCR / qPCR
What is the purpose of G-banding?
Stains condensed chromosomes»_space; visible karyotype.
-heterochromatic (AT-rich) stains dark
What stains are used for G-banding? (2)
- Trypsin and Leishman’s
- Giemsa stain
What stage of the cell cycle are cells paused at for G-banding?
Metaphase.
What is the general process of FISH?
Probe >> fluorescent label >> denatured >> hybridisation >> visualisation (UV).
What types of probes are used for FISH? (3)
- UNIQUE SEQUENCE (» highlight small region, e.g. dup)
- CENTROMERIC (» no. of copies of each chromosome e.g. trisomy)
- PAINTS (» each chromosome different colour e.g. translocation)
What is FISH used for? (5)
- Copy number imbalance
- Aneuploidy
- Confirm G-banding
- Confirm array CGH
- Identify specific abnormalities in cancer
What is copy number variation?
DNA segment with variable copy number to reference genome.
- 12% human genome
- benign / malignant
What is a high copy number of CCL3L1 associated with?
Decreased susceptibility to HIV.
What is a low copy number of FCGR3B associated with?
Increased susceptibility to inflammatory autoimmune disorders.
What cytogenetic methods are used to assess copy number?
- FISH
- MLPA
- Microarray CGH
- Next generation sequencing
- QF-PCR
- qPCR
What is Multiplex Ligation-dependent Probe Amplification (MLPA)?
Multiplex PCR»_space; detects abnormal copy numbers in up to 50 genomic locations.
-alternative to FISH
MLPA analysis - what peak areas are normal / duplication / deletion?
Normal = 1.0 Duplication = 1.35 Deletion = 0.65
What does microarray CGH analyse?
Analyses copy number variants (genomic imbalances) compared to reference.
- highlights imbalances
- can analyse 1000s of oligonulceotides at a time
What is replacing karyotyping as a 1st line test?
Microarray CGH.
What samples are required for microarray CGH? (2)
- 3ml blood in EDTA
- Control DNA (same sex)
How many oligonulceotides are required to determine copy number change in mircroarray CGH?
3 oligonucleotides.
How many genomic disorders can be identified using microarray CGH?
> 40 genomic disorders.
-including learning disabilities and dysmorphic infants
What are the main advantages of microarray CGH? (3)
- Early diagnosis (1st line)
- High resolution
- Greater accuracy of imbalance location / size
What are the main disadvantages of mircoarray CGH? (4)
- Dosage changes only (not balanced rearrangements)
- Low level mosaics not detected
- Non-pathogenic changes detected
- Needs good quality DNA
What is next generation sequencing (NGS) used for?
Generating sequencing libraries by fragmenting genomic DNA.
-divide genome and compare dosage differences to reference DNA
How many base pairs do NGS beads contain?
250 bp.
What does an increased test:control ratio indicate?
Gain.
What does an decreased test:control ratio indicate?
Loss.
What is quantitative fluorescent PCR (QF-PCR)?
PCR amplification of short tandem repeats using fluorescent primers.
-products quantified as peak areas
» compared to reference
What is prenatal aneuploidy detection carried out on?
DNA extracted from amniotic fluid / chorionic villi.
What primers are used for prenatal aneuploidy detection?
Primers from chromosomes 13, 18, 21, X and Y.
What happens in prenatal aneuploidy detection when aneuploidy is present?
2 markers with abnormal dosage.
-size of product directly related to ATTT repeats
QF-PCR; what is a peak area ratio when trisomy is present?
Either 2:1 or 1:1:1.
What is qPCR?
Quantitative / real time PCR.
-confirms small copy number variations
What is qPCR used?
When FISH is unsuitable.
qPCR; what is relative quantitation (RQ)?
Compares difference in concentration between patient sample and normal control.
- 2 different primer sets used
- expressed as a ratio relative to 1
What are the RQ values for normal DNA, deletion and duplication (qPCR)?
Normal - 1.0
Deletion - 1.5
Duplication - 0.5
Which cytogenetic analyses take the least amount of time?
MLPA / QF-PCR.
-10-30 mins per case
Which cytogenetic analysis takes the most amount of time?
G-banding.
-30 mins - 4 hours per case
What are the timescales for the main cytogenetic analyses?
G-banding : 30min - 4hr FISH : 10 min - 1 hr Microarrays : 10 min - 2hr NGS/qPCR : 30 mins - 2hr MLPA/QF-PCR : 10-30 mins
What are the main pre-natal sample types, and when are they carried out? (3)
- Amniocentesis (16w)
- Chorionic villus (12w)
- Non-invasive prenatal testing (12w)
What type of blood sample is used for G-banding?
2-5 ml unclotted.
- T cells stimulated
- Culture 2-3 days
What are the genetic causes of Down syndrome? (3)
- Trisomy 21 (90-95 %)
- Robertsonian Translocation
- Mosaic trisomy 21
What is the recurrence risk of Down syndrome due to trisomy 21?
~1%.
-less if post-zygotic
What is the recurrence risk of Down syndrome due to Robertsonian translocation?
Both parents - 100%.
Maternal - 12%.
Paternal - 2%.
-less in paternal; preselection prevents unbalanced sperm»_space; egg
What is Robertsonian translocation?
Translocation in an acrocentric chromosome.
- 13, 14, 15, 21, 22
- normally t(14;21) in Down
What testing is done to clarify imbalances?
- Array CGH to analyse imbalance
- check if OMIM morbid genes
- FISH to confirm
What are OMIM morbid genes?
Online Mendelian Inheritance in Man.
-lists relationship between genes and phenotypes
What proportion of recurrent miscarriages are due to balanced chromosome changes?
2-3%.
What proportion of infertile men have a chromosome change?
10%.
-usually Klinefelter (47, XXY) or Y deletion
What chromosome change causes female infertility?
Turner syndrome (45, X).
How can t(4;11) cause recurrent miscarriages?
Try to pair the wrong chromosomes.
What is the Pachytene cross?
3rd stage of prophase of meiosis.
- chromosomes divide into 4 chromatids
- 50% chance of unbalanced gamete if translocation
What are the main methods of non-invasive prenatal testing (NIPT)?
- Maternal blood
- Extract free foetal DNA
What is non-invasive prenatal testing used to assess?
Aneuploidy.
-invasive test to confirm
What are the main indicators for prenatal testing? (4)
- Increased maternal age
- Serum screen risk
- Abnormal ultrasound
- FH
What proportion of Down’s births involve mothers >35 years?
40%.
What are the results of a prenatal serum screen (triple test) if the child has Down syndrome?
- Decreased alpha-fp
- Increased oestriol
- Increased hcg
How is amniotic fluid / chorionic villi tested?
DNA extraction
» QF-PCR
» culture cells if abnormal (7-14days)
» G-banding
What proportion of trisomy 21 foetuses spontaneously abort?
25%.
-post 16 weeks
What proportion of spontaneous abortions have a chromosome abnormality, and what tests are carried out?
50%.
- G-banding / QF-PCR / MLPA (aneuploidy)
- array CGH (dosage)
What cytogenetic analysis is carried out for leukaemia?
1ml unclotted bone marow
» culture overnight
» G-banding / FISH
What chromosomal abnormality is associated with leukaemia (CML)?
Philadelphia chromosome.
- t(9;22)
- bcr/abl fusion gene
What cytogenetic analsysis is carried out on solid tumours?
- Fresh tumour»_space; FISH / G-banding
- Archived tissue (paraffin)»_space; FISH / genotyping
What gene is involved in neuroblastoma?
MYCN gene.