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EMS - Genetics in Medicine > Techniques of Chromosomal Analysis (10 & 11) > Flashcards

Techniques of Chromosomal Analysis (10 & 11) Flashcards

1
Q

Whole genome testing

A

G-banding, Next Generation Sequencing and Microarrays

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2
Q

Targeted testing

A

FISH, MLPA, QF-PCR or qPCR

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3
Q

Prenatal diagnosis

A

Amniotic fluid or chorionic villus

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4
Q

Postnatal diagnosis

A

Blood or products of conception

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5
Q

Oncology

A

Solid tumours or leukaemia

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6
Q

Is AT or CG light?

A

CG - higher concentration of genes

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7
Q

Types of probes for FISH

A

Unique sequencing, centromeric and paints

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8
Q

What is a unique sequencing probe in FISH used to see?

A

Deletions or duplicaitons

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9
Q

What is a centromeric probe in FISH used to see?

A

Centromeres - increased condensed repetitive DNA, total number of copy of chromosomes

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10
Q

What is a paint probe in FISH used to see?

A

All genes (translocations), not used anymore

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11
Q

Applications of FISH

A

Aneuploidy, copy number imbalance, confirmation/clarification of G-banding, confirmation of array CGH, identify specific abnormalities in cancer

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12
Q

Copy number variation

A

12% genome, pathogenic/benign, familial/de novo

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13
Q

Copy number leading to disease

A

Autism or acquired cancers

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14
Q

Copy number leading to resistance

A

High copy number of CCL3L1 leads to decrease in susceptibility to HIV

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15
Q

Copy number leading to susceptibility

A

Low copy number of FCGR3B leads to an increase in susceptibility to inflammatory autoimmune disorders

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16
Q

Molecular cytogenetic methods to assess copy number

A

FISH, MLPA, microarray, CGH, next generation sequencing, QF-PCR, qPCR

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17
Q

MLPA

A

Multiplex Ligation-dependent Probe Amplification

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18
Q

What is MLPA?

A

DNA-based, multiplex PCR, detect copy number changes in up to 50 different genomic locations simultaneously, alternative to FISH

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19
Q

Microarray CGH (array CGH)

A

Genome-wide screen, hybridise sample and control DNA to a microarray ‘chip’ with 1000s of DNA spots (oligonuceltoides), higher detection rates

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20
Q

Which method is most important?

A

Microarray CGH - replacing karyotyping as 1st line test

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21
Q

Requirements for Microarray CGH

A

3ml blood in EDTA (also 1-2ml lithium heparin blood for cell culture if follow up needed), control DNA from same sex

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22
Q

why is microarray CGH good?

A

Can look at 8 different patients at a time, each spot contains up to 65,000 spots of DNA in a specific order

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23
Q

Microarray CGH software

A

Allows data from microarray scanner to be read and interpreted, check variants flagged for pathogenicity

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24
Q

Interpreting microarray CGH results

A

Red = loss
Green = gain
Log2 ratio 0.3

25
Q

Determining regions for potential copy number change

A

At least 3 oligonucleotides required, call imbalances >150,000 bases, database searches to ascertain pathogenicity of imbalances

26
Q

Applications of Array CGH

A

Moderate to severe learning and development disability (LDD) 1-3% (IQ 40 genomic disorders

27
Q

Advantages of array CGH

A
  • Early diagnosis (1st line test)
  • High resolution (high diagnostic hit rate)
  • Greater accuracy of location/size of imbalances
  • Info on relevant genes
28
Q

Disadvantages of array CGH

A
  • Dosage change only (not mutations/balanced rearrangements)
  • Low level mosaics not detected
  • Non-pathogenic and uncertain pathogenic changes detected
  • Needs good quality DNA
29
Q

Future technologies

A

Analyse mutations and dosage changes e.g. exomes and whole genomes

30
Q

Next generation sequencing

A

Look at 10-20 patients, molecular barcode rather than fluorescent, sequencing libraries generated by fragmenting genomic DNA and adaptor ligation, flow cell 20-40 patients

31
Q

Analysis of next generation sequencing

A

Divide into digital windows, analyse dosage differences in patients against reference, 80 reads/window, increase test:control = gain

32
Q

QF-PCR

A

PCR amplification of short tandem repeats using fluorescent primers

33
Q

How is QF-PCR visualised?

A

Peak areas using automated DNA sequencer, assess relative amount of DNA against control

34
Q

Prenatal aneuploidy detection

A

DNA extraction from amniotic fluid/chorionic villi, PCR amplification for up to 4/5 markers (13, 18, 21, X and Y), aneuploidy > 2 markers with abnormal dosage

35
Q

Micro satellite tetra nucleotide repeat markers

A

PCR of a tetranucelotide repeat analysed on fluorescent sequencer, uses a polyacrylamide gel to separate products by size, 2 copies of an allele = peak 2 x size

36
Q

qPCR

A

Confirms small CNVS, when FISH is unsuitable, expressed as ratio relative to 1, 0.5 deletion, 1.5 duplication

37
Q

How long does G-banding take?

A

30 mins-4 hours/case

38
Q

How long does FISH take?

A

10 mins-1 hour/case

39
Q

How long do Microarrays take?

A

10 mins- 2 hours/case

40
Q

How long does NGS/qPCR take?

A

30 mins-2 hours/case

41
Q

How long does MLPA/QF-PCR take?

A

10 mins-30 mins/case

42
Q

Samples of cytogenetics

A

Blood, amniotic fluid, placenta, other foetal tissue, bone marrow or tumour

43
Q

Referrals for blood cytogenetic studies

A

Dysmorphic newborns, gender assignment, developmental problems (intellectual, physical, sexual), heart defect, reproductive problems, family studies

44
Q

Blood for G-banding

A

2-5ml unclotted (heparin), stimulate T-lymphocytes, cultures 2-3 days, analysis 30 mins - 4 hours

45
Q

Different ways Downs syndrome can be inherited?

A

Free trisomy 21 (90-95%), Robertsonian translocation (2-5%), Mosaic trisomy 21 (2-5%)

46
Q

Robertsonian translocation

A

46XX der (21;21)(q10;q10) 100% recurrence risk

46XX her (14;21)(q10;q10)+ 21 
male carrier 2% recurrence risk, female 12%
47
Q

Prenatal diagnosis

A

Amniocentesis (16 wks), Chorionic villus biopsy (12 wks), non-invasive prenatal testing (12 wks)

48
Q

NIPT

A

Maternal blood/free circulating fetal DNA, assess aneuploidy 13, 18, 21, next generation sequencing, risk invasive test to confirm

49
Q

Indication of aneuploidy pre-natal

A

Increased maternal age, serum screen risk, abnormal ultrasound scan (USS), family history, previous chromosomal abnormality

50
Q

What percentage of trisomy 21 spontaneously abort post 16 weeks?

51
Q

What percentage of pregnancies is the mother >35, and what percentage of these babies have Down’s syndrome?

A

15% of all pregnancies above >35, 40% Downs

52
Q

Cytogenetics and amniotic fluid

A
  • Portion for DNA extraction (QF-PCR)
  • Separate cells from remaining fluid
  • Culture cells (7-14 days) if QF-PCR abnormal
  • G-banded analysis
53
Q

Cytogenetics and chorionic villi

A
  • Separate maternal and foetal tissue
  • QF-PCR
  • Culture cells (7-14 days) if QF-PCR abnormal
  • G-banded analysis
54
Q

Spontaneous abortion

A

50% chromsome abnormaltiy (triploidy, 45X, trisomy), tissues of skin, placenta, lung cartilage, cord

55
Q

Investigating spontaneous abortion

A

Macerate tissue, either cell culture (7-28 days) and G-banding or QF-PCR/MLPA to assess aneuploidy, or array CGH (extracted DNA) to assess dosage

56
Q

Preimplantation genetic analysis

57
Q

Cytogenetics and cancer

A

Usually translocation and dosage change

58
Q

Cytogenetics and Leukaemia

A
  • 1ml unclotted bone marrow, suspension culture overnight, G-banded analysis/FISH
  • Chromosome poor quality
  • Philadelphia translocation t(9;22) in CML (ABL and BCR fuse)
59
Q

Cytogenetics and solid tumour genetics

A

Use tumour tissue

  • Fresh tumour - FISH/G-banding (culture 1-20 days)
  • Archived tissue (paraffin embedded) - FISH/genotyping
  • MYCN neuroblastoma - poor prognosis and increase dose of chemo

EMS - Genetics in Medicine (14 decks)

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