Numerical Chromosomal Abnormalities Flashcards

1
Q

What are histones?

A

o Histones are highly positively charged proteins that are attracted to the negative charge of DNA. Imagine this like coiling a garden hose up; in takes up less room to store than if you leave the garden hose out stretched. But it requires energy to coil that hose up and essentially that is what histones supply. They give the DNA a support to wrap around
A chromosome is an organized package of DNA found in the nucleus of the cell. Different organisms have different numbers of chromosomes. Humans have 23 pairs of chromosomes–22 pairs of numbered chromosomes, called autosomes, and one pair of sex chromosomes, X and Y. Each parent contributes one chromosome to each pair so that offspring get half of their chromosomes from their mother and half from their father.

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2
Q

Describe the structure of a chromosome

What is euchomatin and hetrochromatin?

How is DNA usually packed?

What is chromatin?

A

o Chromosomes usually exists as chromatin
- DNA double helix bounds to histones
- Octamer of histones form nucleosome
o Euchromatin – open
- Extended state, dispersed through nucleus
- Allows gene expression
o Heterochormatin – highly condensed
- Highly condensed, genes not expressed
o DNA usually loosely packed, except during cell division when DNA is complexed with various proteins and undergoes several levels of compaction through coiling and supercoiling.
o Nucleosome is fundamental unit of DNA – eight histones and two turns of DNA
o DNA + Proteins = Chromatins
o Exist in homologous pairs
o Two copies of each chromosome  maternal and paternal copies
o The homologues have the same genes on them (the autosomes) but they may have different allelic forms
o Same loci on each chromosome.

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3
Q

Why are chromosomes sometimes shown with a single chromatid?

A

Like in interphase

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4
Q

• Why are chromosomes sometimes shown with two sister chromatids?

A

Replication

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5
Q

Describe the phases of the cell cycle

A

o G1 - Cell makes a variety of proteins needed for DNA replication
o S - synthesis; chromosomes are replicated so that each chromosome now consists of two sister, identical chromatids
o G2 - synthesis of proteins especially microtubules, error checks wrt the chromosomes.
o Some cells don’t replicate; some are senescent.
o Our cells are mostly quiescent  single chromatids at this point
o The cell cycle only begins when we need new cells
o S phase is where DNA duplicates so there are exact copies of the single chromatids
 Go from a single chromatid to two identical chromatids being attached
 This is necessary to produce the daughter cells

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6
Q

What is metacentric, submetacentric and acrocentric

A
o	Metacentric 
-	p & q arms even length 
-	1-3, 16-18
o	Submetacentric
-	p arm shorter than q
-	4-12, 19-20, X
o	Acrocentric
-	Long q, small p 
-	p contains no unique DNA
-	13-15, 21-22, Y
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7
Q

What chromosomal changes can happen and how are they detected?

A

o Numerical – can be detected through traditional karyotyping, FISH, QF-PCR, NGS
o Structural - can detect through traditional karyotyping, FISH
o These techniques are good for visualising big changes to the genome – and those big changes fall into these two categories.
o Numerical and structural.

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8
Q

An give me an example of a numerical change?

A

Down’s syndrome

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9
Q

What’s the genetic change which causes Down’s syndrome?

A

Trisomy 21

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10
Q

Define haploid, diploid, polyploid and aneuploid

A

o HAPLOID - one set of chromosomes (n=23) as in a normal gamete.
o DIPLOID - somatic cells, cell contains two sets of chromosomes (2n=46; normal in human)
o POLYPLOID - extra sets of chromosomes  rare
- multiple of the haploid number (e.g. 4n=92)
- STERILE
o ANEUPLOID - chromosome number which is not an exact multiple of haploid number - due to extra or missing chromosome(s) (e.g. 2n+1=47 or -1=45)
- Downs, Patau, Edward syndrome
o Trisomy
o Monosomy
o Mosaicism – a mixed collection of cells. Some could be trisomic others normal

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11
Q

Have a look at mitosis and meiosis

A

On image

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12
Q

How does aneuploidy arise?

A

o If non-disjunction occurs in meiosis I  the two homologues both go into a single cell. So you end up with two copies of the chromosome in the same cell and none in another
o In Meiosis II  disjunction happens appropriately and pulls the chromatids apart and distributes them into the resulting gametes  but we now have two copies of that chromosome in that gamete  known as being DISOMIC
o No copies  NULLISOMIC
o Alternatively we could see that disjunction happens appropriately in meiosis I  but then in meiosis II two of the single chromatids have ended up in the same gamete  DISOMIC
o Gametes should be MONOSOMIC

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13
Q

Describe non-disjunction

A

Pulling apart chromosomes at centromere= disjunction. Improper disjunction= nondisjunction (e.g. trisomy, monosomy). Main cause of aneuploidy.

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14
Q

What is mosaicism?

What causes it?

What is anaphase lag?

A
  • Presence of two or more genetically different cell lines derived from a single zygote. E.g. disomy 21, trisomy 21 and monosomy 21- mosaic blastocyte.
  • Mechanisms- post-zygote non-disjunction (mitotic non-disjunction after fertilisation).
  • Anaphase lag- where one homologous chromosome in meiosis or one chromatid in mitosis fails to connect to the spindle apparatus or is tardily drawn to its pole and fails to be included in the reforming nucleus. Forms micronucleus in cytoplasm and lost from cell.
  • The lagging chromosome is not incorporated into the nucleus of one of the daughter cells, resulting in one normal daughter cell and one with monosomy.
  • Common cause of aneuploidy and mosaicism.
  • Can result in trisomic rescue if daughter cell was originally trisomy.
  • Clinical relevance- less severe. Difficult to assess due to proportions and tissues affected. E.g. Down’s/Klinefelter’s/Turner’s.
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15
Q

Define Monosomy

A

o Autosomal are very very rare, found one case report from 1967
o Relatively common sex chromosome monosomy = Turner’s
o Full monosomy arise by NDJ
o Partial monosomy (microdeletion syndromes) far more common – mechanism different
- Only a chunk of the second X chromosome is missing

  • Rarely autosomal.
  • Common sex chromosome monosomy is Turner’s syndrome.
  • Full monosomy arising from nondisjunction.
  • Partial monosomy far more common, due to microdeletions.
  • Nullisomic gametes (no X) fertilised with Y chromosome is lethal- YO. XO is not lethal- female presenting.
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16
Q

How does Turner’s (45,X) arise?

A

o Blue = Maternal sex chromosome; black = paternal sex chromosome
o Nullisomic gametes fertilised with a sperm carrying an X chromosome will be XO (Turners)
o Nullisomic gametes fertilised with a sperm carrying a Y chromosome will be YO
o Disomic gametes can be

17
Q

What is Prenatal Diagnosis – Chorionic Villus Sampling?

A

o 11-14 weeks
o Miscarriage rate 0.5% to 1%
o Maternal contamination
o Transverse limb defects

18
Q

What is Prenatal Diagnosis – Amniocentesis?

A

o >16 weeks
o Extraction of amniotic fluid
o Biochemical diagnosis possible
o Miscarriage risk (0.5-1%)

19
Q

What is G-banding?

A
o	Giemsa stain
o	Metaphase
o	Line-up based on
-	Size
-	Banding
-	Centromere position
o	Giemsa highlights heterochromatic regions which are less likely to contain genes.  But the crucial thing is that the banding can be used to differentiate between chromosomes ant to compare chromosomes.
20
Q

Do heterochromatin and euchromatin stain the same?

A

 Heterochromatic and euchromatin stain differently
 Euchromatin = GC-rich; loosely packed; genes active. Lighter.
 Heterochromatin = AT-rich; tightly packed; genes inactive. Darker

21
Q

What is FISH?

A

FISH - Fluorescent in situ hybridisation
o Very effective to look for numerical and structural abnormalities.
o Cultured cells, metaphase spread
o Microscopic (5-10Mb)
1) Fluorescent probe
2) Denature probe and target DNA
3) Mix probe and target DNA
4) Probe binds to target
o It uses cultured cells, this takes longer than QF-PCR
o Use FISH to design probes which are specific to certain chromosomes
o Resolution – how big or small an abnormality can be detected
o G banding good at detecting big deletion events, not small subtle events
o Probe = fluorophore
o Fluorescence in situ hybridization (FISH) is a molecular diagnostic technique utilizing labeled DNA probes to detect or confirm gene or chromosome abnormalities. It is often used in cancer diagnosis. The sample DNA (metaphase chromosomes or interphase nuclei) is first denatured, a fluorescently labeled probe of interest is then added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it re-anneals back into a double stranded DNA. The probe signal can then be seen through a fluorescent microscope and the sample DNA can be scored for the presence or absence of the signal. Unlike most other techniques used to study chromosomes, FISH does not have to be performed on cells that are actively dividing. This makes it a very versatile procedure. Uses encompass a wide range of applications such as the detection of aneuploidy, constitutional microdeletion syndromes as well as rearrangements. These aberrations have clinical implications for numerous genetic diseases such as leukemia, lymphoma, solid tumors, autism and other developmental syndromes. FISH probes are commonly made from BAC clones. Empire Genomics provides access to over one million BAC clones from the Roswell Park Cancer Institute genomic libraries, which are available for purchase as either unlabeled or custom labeled FISH probes. Empire Genomics can provide custom designed FISH probes and also offers a number of premade gene specific probes. Control probes are available as well.
o Denaturing the probe and patient DNA, the probe will preferentially bind to the DNA, integrated into the patients genome. Under uv light the probe will glow on the metaphase chromosome
o good at detecting big deletion events, not small subtle events

22
Q

Describe Quantitative fluorescence PCR

A

o Design primers for a specific microsatellite that is on chromosome 21 and amplify up the region using PCR and then look to see how big the microsatellite regions are and how many copies there are
o FISH and G-banding take days so this is much quicker, no culturing involved,
o Microsatellite typically not associated with disease, normal variations
o 3 copies of chromosomes 21 in that individual different size alleles
o 2 alleles same size and position refers to 2 copies.
o Quick test for trisomy, have to have a suspicison e.g. trisonmic 13 or 21.

23
Q

What are the types of prenatal tests?

A

o Invasive – classic techniques
- Amniocentesis (14-20 wks, amniotic fluid)
- Chorionic villus sampling (CVS) (11-14 wks, placental cells)
o Non-invasive – enables foetal DNA to be isolated
- Cell free foetal DNA (cffDNA): DNA fragments in maternal plasma (10 wks onwards)
- Actually for trisomies still need confirmation with amnio/CVS
- Reducing no of women to give an unnecessary proc to
o Cff dna =- extracting dna from maternal plasma 14:40 mins

24
Q

What is Prenatal Diagnosis – Non Invasive Diagnosis?

A

o SAFE TEST – Trisomies 13, 18, 21
o Available privately ~ £400
o Can sequence the entire foetal genome and can see if there are the same number of copies
o But if a foetus is trisomic you will have extra copies of that chromosome
o A quick and non-invasive way to detect trisomy
o Use standrad pcr to amp a specific gene e//g monogenic disorder
o Due to a de novo mutation = amplify spec the dna
o Chromosomal abnormalities using NGS capture foetal DNA, and get a read/stretch of dna along genome.
o 50 frags at every pos…. At trisomy a lot my fragments

25
Q

What is downs syndrome?

A

On document

26
Q

What is Patau Syndrome – Trisomy 13?

A

On document

27
Q

What is Edwards syndrome - Trisomy 18?

A

On document

28
Q

What is turner syndrome?

A

On document

29
Q

What is Klinefelter (47,XXY)?

A

On document