Structural Chromosomal Abnormalaties Flashcards

1
Q

What are some chromosomal structural abnormalities

A

Translocation:

  • Reciprocal
  • Robertsonian

Inversion
Deletion
Duplication
Rings
Isochromosomes

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2
Q

What is translocation

A
  • Most common
  • Exchange of two segments between non-homologous chromosomes
  • Reciprocal or Robertsonian
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3
Q

How does translocation due to inappropriate NHEJ work

A
  • Incorrectly sticks 2 ends of non-homologous chromosomes together.
  • creates derivatives
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4
Q

how are unbalanced individuals produced?

A
  • Chromosomes align normally as a bivalent structure but here it forms a tetravalent structure and separates either way.
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5
Q

What is the result of unbalanced reciprocal translocation

A
  • Many lead to miscarriage (hence why a woman with a high number of unexplained miscarriages should be screened for a balanced translocation)
  • Learning difficulties, physical disabilities
  • Tend to be specific to each individual so exact risks and clinical features vary
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6
Q

Explain what happens in robertsonian translocation

A
  • The combination of Q arms of 2 chromosomes
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7
Q

What are the characteristics of Robertsonian translocations

A
  • Two acrocentric chromosomes join near centromere with the loss of p arms
  • Balanced carrier has 45 chromosomes
  • If 46 chromosomes present including Robertsonian then must be unbalanced
  • p arms encode rRNA (multiple copies so not deleterious to lose some)
  • Robertsonian translocations 13;14 and 14;21 relatively common. 21;21 translocation leads to 100% risk of Down syndrome in fetus
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8
Q

What are some outcomes of translocations?

A
  • Very difficult to predict
  • Only have approximate probability of producing possible gametes
  • Some unbalanced outcomes may lead to spontaneous abortion of conceptus so early that not seen as problem
  • Some unbalanced outcomes may lead to miscarriage later on and present clinically
  • Some may result in live-born baby with various problems
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9
Q

List some other structural changes

A
  • Terminal deletion- the end of a chromosome is deleted
  • Interstitial deletion - the middle of a chromosome is taken out and resealed
  • Inversion - The middle of a chromosome is inverted and resealed
  • Duplication- a portion of the chromosome is duplicated and extends the chromosomes
  • Ring Chromosome - The chromosomes sealed into a ring shape
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10
Q

What are some characteristics of deletion mutations?

A
  • 1:7000 live births
  • Deletion may be terminal or interstitial
  • Causes a region of monosomy
  • Haploinsufficiency of some genes
  • Contiguous gene syndrome (multiple, unrelated clinical features)
  • Phenotype is specific for size and place on deletion
  • Gross deletions seen on metaphase spread on G-banded karyotype
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11
Q

What are some characteristics of microdeletion mutations?

A
  • Many patients had no abnormality visible on metaphase spread
  • High resolution banding, FISH and now CGH showed ‘micro’ deletions
  • Only a few genes may be lost or gained
    -Velocardiofacial (DiGeorge), 22q11
    Wolf-Hirschhorn, 4p16
  • Williams, 7q11
  • Smith-Magenis, 17p11
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12
Q

Describe unequal crossing-over

A
  • When homologous chromosomes don’t align equally
  • Leads to genes not crossing over on the right locus
  • Changes the length of the chromosomes
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13
Q

How and where do we acquire samples for testing

A

Prenatal:

  • Amniocentesis, amniotic fluid
  • Chorionic villus sampling, Placenta
  • Cell-free fetal DNA, Maternal plasma

Postnatal:

  • Blood
  • Saliva
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14
Q

How does chromosome staining work

A

Most common = G-banding
G = Giemsa

Why bands?

  • Chromatin
  • 2 different sorts: euchromatin & heterochromatin
  • Euchromatin = GC-rich; loosely packed; genes active
  • Heterochromatin = AT-rich; tightly packed; genes inactive
  • Stain differently
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15
Q

What are the steps in analysing a blood sample

A
  • Draw 5ml of venous blood
  • Add phytohemagglutinin and culture medium
  • Culture at 37C for 3 days
  • Add colchicine and hypotonic saline
  • Cells fixed
  • Spread cells onto the slide by dropping
  • Digest with trypsin and stain with Giernsa
  • Analyse metaphase spread
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16
Q

Summarise the G-banding technique

A
  • How does karyotype of patient differ from expected?
  • Uses a chemical stain
  • Uses metaphase chromosomes
  • Takes several days at least
  • Looks for aneuploidies, translocations & very large deletions
17
Q

What is FISH

A

Fluorescent in situ hybridisation

Hybridisation = single stranded nucleic acid strand binds to a new single stranded nucleic acid strand (DNA/DNA or DNA/RNA)

Cultured cells, metaphase spread:

  • Fluorescent probe
  • Denature probe and target DNA
  • Mix probe and target DNA
  • Probe binds to target
18
Q

What is a probe?

A
  • A single stranded DNA (or RNA) molecule
  • Typically 20 – 1000 bases in length
  • Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
  • In some techniques thousands or millions of probes are used simultaneously
19
Q

What is ArrayCGH

A
  • Similar to microarray technologies
  • Array comparative genomic hybridisation
  • For detection of sub-microscopic chromosomal abnormalities
  • Patient DNA labelled Green
  • Control DNA labelled Red
20
Q

What is QF-PCR

A
  • Quantitative fluorescence polymerase chain reaction
  • Trisomies 13, 18 and 21
  • Uses microsatellites
21
Q

What are micro satellites

A
  • Short repeated sequences
  • Number of repeats varies between individuals
  • Total length of microsatellite sequence varies between individuals
22
Q

How can we detect micro satellites

A
  • Isolate DNA from individual
  • Design primers specific to flanking sequences
  • PCR amplification
  • Gel electrophoresis
23
Q

How does PCR work

A

Exponential amplification of a DNA fragment of known sequence

PCR consists of incubating at three different temperatures

This results in three different processes happening:

  • Denaturation
  • Annealing
  • Extension
24
Q

How is QF-PCR used to detect chromosomal abnormalities

A
  • Perform PCR using primers for microsatellite known to be on chromosome 21 (if testing for Down’s)
  • Should be two copies of microsatellite (one from mother, one from father, like any other autosomal locus, gene, whatever)
  • If homozygous, there will be a single peak of high signal
  • If heterozygous, there will be two peaks of similar, lower signal
25
Q

What is Non-invasive pre-natal testing and NGS

A
  • Cell free fetal DNA
  • Maternal blood sample
  • Trisomy testing
  • Next-generation sequencing
  • “High chance” indicator for invasive test