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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
Determining regions for potential copy number change
At least 3 oligonucleotides required, call imbalances >150,000 bases, database searches to ascertain pathogenicity of imbalances
26
Applications of Array CGH
Moderate to severe learning and development disability (LDD) 1-3% (IQ 40 genomic disorders
27
Advantages of array CGH
- Early diagnosis (1st line test) - High resolution (high diagnostic hit rate) - Greater accuracy of location/size of imbalances - Info on relevant genes
28
Disadvantages of array CGH
- Dosage change only (not mutations/balanced rearrangements) - Low level mosaics not detected - Non-pathogenic and uncertain pathogenic changes detected - Needs good quality DNA
29
Future technologies
Analyse mutations and dosage changes e.g. exomes and whole genomes
30
Next generation sequencing
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
Analysis of next generation sequencing
Divide into digital windows, analyse dosage differences in patients against reference, 80 reads/window, increase test:control = gain
32
QF-PCR
PCR amplification of short tandem repeats using fluorescent primers
33
How is QF-PCR visualised?
Peak areas using automated DNA sequencer, assess relative amount of DNA against control
34
Prenatal aneuploidy detection
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
Micro satellite tetra nucleotide repeat markers
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
qPCR
Confirms small CNVS, when FISH is unsuitable, expressed as ratio relative to 1, 0.5 deletion, 1.5 duplication
37
How long does G-banding take?
30 mins-4 hours/case
38
How long does FISH take?
10 mins-1 hour/case
39
How long do Microarrays take?
10 mins- 2 hours/case
40
How long does NGS/qPCR take?
30 mins-2 hours/case
41
How long does MLPA/QF-PCR take?
10 mins-30 mins/case
42
Samples of cytogenetics
Blood, amniotic fluid, placenta, other foetal tissue, bone marrow or tumour
43
Referrals for blood cytogenetic studies
Dysmorphic newborns, gender assignment, developmental problems (intellectual, physical, sexual), heart defect, reproductive problems, family studies
44
Blood for G-banding
2-5ml unclotted (heparin), stimulate T-lymphocytes, cultures 2-3 days, analysis 30 mins - 4 hours
45
Different ways Downs syndrome can be inherited?
Free trisomy 21 (90-95%), Robertsonian translocation (2-5%), Mosaic trisomy 21 (2-5%)
46
Robertsonian translocation
46XX der (21;21)(q10;q10) 100% recurrence risk ``` 46XX her (14;21)(q10;q10)+ 21 male carrier 2% recurrence risk, female 12% ```
47
Prenatal diagnosis
Amniocentesis (16 wks), Chorionic villus biopsy (12 wks), non-invasive prenatal testing (12 wks)
48
NIPT
Maternal blood/free circulating fetal DNA, assess aneuploidy 13, 18, 21, next generation sequencing, risk invasive test to confirm
49
Indication of aneuploidy pre-natal
Increased maternal age, serum screen risk, abnormal ultrasound scan (USS), family history, previous chromosomal abnormality
50
What percentage of trisomy 21 spontaneously abort post 16 weeks?
25%
51
What percentage of pregnancies is the mother >35, and what percentage of these babies have Down's syndrome?
15% of all pregnancies above >35, 40% Downs
52
Cytogenetics and amniotic fluid
- Portion for DNA extraction (QF-PCR) - Separate cells from remaining fluid - Culture cells (7-14 days) if QF-PCR abnormal - G-banded analysis
53
Cytogenetics and chorionic villi
- Separate maternal and foetal tissue - QF-PCR - Culture cells (7-14 days) if QF-PCR abnormal - G-banded analysis
54
Spontaneous abortion
50% chromsome abnormaltiy (triploidy, 45X, trisomy), tissues of skin, placenta, lung cartilage, cord
55
Investigating spontaneous abortion
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
Preimplantation genetic analysis
QF-PCR
57
Cytogenetics and cancer
Usually translocation and dosage change
58
Cytogenetics and Leukaemia
- 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
Cytogenetics and solid tumour genetics
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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