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Year 2 EMS GiM > Cytogenetics - Techniques of Analysis > Flashcards

Cytogenetics - Techniques of Analysis Flashcards

1
Q

What techniques are used to sequence the whole genome? (3)

A
  • G banding
  • Next generation sequencing
  • Microarrays
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2
Q

What are the main targeted tests? (3)

A
  • FISH
  • MLPA
  • QF-PCR / qPCR
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3
Q

What is the purpose of G-banding?

A

Stains condensed chromosomes&raquo_space; visible karyotype.

-heterochromatic (AT-rich) stains dark

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4
Q

What stains are used for G-banding? (2)

A
  • Trypsin and Leishman’s

- Giemsa stain

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5
Q

What stage of the cell cycle are cells paused at for G-banding?

A

Metaphase.

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6
Q

What is the general process of FISH?

A 
Probe
>> fluorescent label
>> denatured
>> hybridisation
>> visualisation (UV).
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7
Q

What types of probes are used for FISH? (3)

A
  • 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)
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8
Q

What is FISH used for? (5)

A
  • Copy number imbalance
  • Aneuploidy
  • Confirm G-banding
  • Confirm array CGH
  • Identify specific abnormalities in cancer
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9
Q

What is copy number variation?

A

DNA segment with variable copy number to reference genome.

  • 12% human genome
  • benign / malignant
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10
Q

What is a high copy number of CCL3L1 associated with?

A

Decreased susceptibility to HIV.

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11
Q

What is a low copy number of FCGR3B associated with?

A

Increased susceptibility to inflammatory autoimmune disorders.

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12
Q

What cytogenetic methods are used to assess copy number?

A
  • FISH
  • MLPA
  • Microarray CGH
  • Next generation sequencing
  • QF-PCR
  • qPCR
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13
Q

What is Multiplex Ligation-dependent Probe Amplification (MLPA)?

A

Multiplex PCR&raquo_space; detects abnormal copy numbers in up to 50 genomic locations.
-alternative to FISH

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14
Q

MLPA analysis - what peak areas are normal / duplication / deletion?

A 
Normal = 1.0
Duplication = 1.35
Deletion = 0.65
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15
Q

What does microarray CGH analyse?

A

Analyses copy number variants (genomic imbalances) compared to reference.

  • highlights imbalances
  • can analyse 1000s of oligonulceotides at a time
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16
Q

What is replacing karyotyping as a 1st line test?

A

Microarray CGH.

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17
Q

What samples are required for microarray CGH? (2)

A
  • 3ml blood in EDTA

- Control DNA (same sex)

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18
Q

How many oligonulceotides are required to determine copy number change in mircroarray CGH?

A

3 oligonucleotides.

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19
Q

How many genomic disorders can be identified using microarray CGH?

A

> 40 genomic disorders.

-including learning disabilities and dysmorphic infants

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20
Q

What are the main advantages of microarray CGH? (3)

A
  • Early diagnosis (1st line)
  • High resolution
  • Greater accuracy of imbalance location / size
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21
Q

What are the main disadvantages of mircoarray CGH? (4)

A
  • Dosage changes only (not balanced rearrangements)
  • Low level mosaics not detected
  • Non-pathogenic changes detected
  • Needs good quality DNA
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22
Q

What is next generation sequencing (NGS) used for?

A

Generating sequencing libraries by fragmenting genomic DNA.

-divide genome and compare dosage differences to reference DNA

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23
Q

How many base pairs do NGS beads contain?

A

250 bp.

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24
Q

What does an increased test:control ratio indicate?

A

Gain.

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25
Q

What does an decreased test:control ratio indicate?

A

Loss.

26
Q

What is quantitative fluorescent PCR (QF-PCR)?

A

PCR amplification of short tandem repeats using fluorescent primers.
-products quantified as peak areas
» compared to reference

27
Q

What is prenatal aneuploidy detection carried out on?

A

DNA extracted from amniotic fluid / chorionic villi.

28
Q

What primers are used for prenatal aneuploidy detection?

A

Primers from chromosomes 13, 18, 21, X and Y.

29
Q

What happens in prenatal aneuploidy detection when aneuploidy is present?

A

2 markers with abnormal dosage.

-size of product directly related to ATTT repeats

30
Q

QF-PCR; what is a peak area ratio when trisomy is present?

A

Either 2:1 or 1:1:1.

31
Q

What is qPCR?

A

Quantitative / real time PCR.

-confirms small copy number variations

32
Q

What is qPCR used?

A

When FISH is unsuitable.

33
Q

qPCR; what is relative quantitation (RQ)?

A

Compares difference in concentration between patient sample and normal control.

  • 2 different primer sets used
  • expressed as a ratio relative to 1
34
Q

What are the RQ values for normal DNA, deletion and duplication (qPCR)?

A

Normal - 1.0
Deletion - 1.5
Duplication - 0.5

35
Q

Which cytogenetic analyses take the least amount of time?

A

MLPA / QF-PCR.

-10-30 mins per case

36
Q

Which cytogenetic analysis takes the most amount of time?

A

G-banding.

-30 mins - 4 hours per case

37
Q

What are the timescales for the main cytogenetic analyses?

A 
G-banding : 30min - 4hr
FISH : 10 min - 1 hr
Microarrays : 10 min - 2hr
NGS/qPCR : 30 mins - 2hr
MLPA/QF-PCR : 10-30 mins
38
Q

What are the main pre-natal sample types, and when are they carried out? (3)

A
  • Amniocentesis (16w)
  • Chorionic villus (12w)
  • Non-invasive prenatal testing (12w)
39
Q

What type of blood sample is used for G-banding?

A

2-5 ml unclotted.

  • T cells stimulated
  • Culture 2-3 days
40
Q

What are the genetic causes of Down syndrome? (3)

A
  • Trisomy 21 (90-95 %)
  • Robertsonian Translocation
  • Mosaic trisomy 21
41
Q

What is the recurrence risk of Down syndrome due to trisomy 21?

A

~1%.

-less if post-zygotic

42
Q

What is the recurrence risk of Down syndrome due to Robertsonian translocation?

A

Both parents - 100%.
Maternal - 12%.
Paternal - 2%.

-less in paternal; preselection prevents unbalanced sperm&raquo_space; egg

43
Q

What is Robertsonian translocation?

A

Translocation in an acrocentric chromosome.

  • 13, 14, 15, 21, 22
  • normally t(14;21) in Down
44
Q

What testing is done to clarify imbalances?

A
  • Array CGH to analyse imbalance
  • check if OMIM morbid genes
  • FISH to confirm
45
Q

What are OMIM morbid genes?

A

Online Mendelian Inheritance in Man.

-lists relationship between genes and phenotypes

46
Q

What proportion of recurrent miscarriages are due to balanced chromosome changes?

A

2-3%.

47
Q

What proportion of infertile men have a chromosome change?

A

10%.

-usually Klinefelter (47, XXY) or Y deletion

48
Q

What chromosome change causes female infertility?

A

Turner syndrome (45, X).

49
Q

How can t(4;11) cause recurrent miscarriages?

A

Try to pair the wrong chromosomes.

50
Q

What is the Pachytene cross?

A

3rd stage of prophase of meiosis.

  • chromosomes divide into 4 chromatids
  • 50% chance of unbalanced gamete if translocation
51
Q

What are the main methods of non-invasive prenatal testing (NIPT)?

A
  • Maternal blood

- Extract free foetal DNA

52
Q

What is non-invasive prenatal testing used to assess?

A

Aneuploidy.

-invasive test to confirm

53
Q

What are the main indicators for prenatal testing? (4)

A
  • Increased maternal age
  • Serum screen risk
  • Abnormal ultrasound
  • FH
54
Q

What proportion of Down’s births involve mothers >35 years?

A

40%.

55
Q

What are the results of a prenatal serum screen (triple test) if the child has Down syndrome?

A
  • Decreased alpha-fp
  • Increased oestriol
  • Increased hcg
56
Q

How is amniotic fluid / chorionic villi tested?

A

DNA extraction
» QF-PCR
» culture cells if abnormal (7-14days)
» G-banding

57
Q

What proportion of trisomy 21 foetuses spontaneously abort?

A

25%.

-post 16 weeks

58
Q

What proportion of spontaneous abortions have a chromosome abnormality, and what tests are carried out?

A

50%.

  • G-banding / QF-PCR / MLPA (aneuploidy)
  • array CGH (dosage)
59
Q

What cytogenetic analysis is carried out for leukaemia?

A

1ml unclotted bone marow
» culture overnight
» G-banding / FISH

60
Q

What chromosomal abnormality is associated with leukaemia (CML)?

A

Philadelphia chromosome.

  • t(9;22)
  • bcr/abl fusion gene
61
Q

What cytogenetic analsysis is carried out on solid tumours?

A
  • Fresh tumour&raquo_space; FISH / G-banding

- Archived tissue (paraffin)&raquo_space; FISH / genotyping

62
Q

What gene is involved in neuroblastoma?

A

MYCN gene.

Year 2 EMS GiM (12 decks)

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