Clinical Genetics: Chromosomal abnormalities I Flashcards
What form of DNA do chromosomes usually exist as?
- Chromosomes usually exists as chromatin
- DNA double helix wound around an octamer of histone proteins
- Octamer of histones form nucleosome
- Nucleosomes packaged together with scaffolding proteins to form chromatin
What is the difference between euchromatin and heterochromatin?
-
Euchromatin
- Uncondensed, dispersed through nucleus
- Allows gene expression
-
Heterochromatin
- Highly condensed, genes not expressed
DNA is usually loosely packaged within the chromosome. When is this not the case?
- Not the case during cell division when DNA is complexed with various proteins and undergoes several levels of compaction through coiling and supercoiling
What are homologous chromosomes?
- Homologous chromosomes are a pair of identical chromosomes, same length, genes and centromere position.
- One of the pair of chromosomes is inherited from your mother and the other inherited from your father
What is a gene locus?
- A gene locus is the location of a particular gene on a chromosome
What is an allele?
- An allele is an alternate form of a gene
- At each gene locus an individual has 2 alleles, one from each homologous chromosome
Why are chromosomes sometimes shown with a single chromatid?
- Chromosomes with single chromatid show how chromosomes look during interphase - after cell division
Why are chromosomes sometimes shown with two sister chromatids?
- Chromosomes with 2 sister chromatids show how chromosomes look after S phase where DNA is duplicated in anticipation of cell division
Briefly describe the stages of the cell cycle
- G1 - Cellular contents, except chromosomes are duplicated, Cell makes proteins needed for DNA replication
- S phase - Chromosomes are replicated so that each chromosome now consists of two sister, identical chromatids
- G2 - Synthesis of proteins especially microtubules
- Mitosis - Cell divison
How many pairs of chromosomes do humnas have?
- 23 pairs of chromosomes
- 22 pairs autosomes, 1 pair sex chromosomes XX or XY
What are the 3 different types of chromosome? State which chromosomes within the human genome belong to each type of chromosome
-
Metacentric - p & q arms even length
- 1-3, 16-18
-
Submetacentric - p arm shorter than q
- 4-12, 19-20, X
-
Acrocentric - Long q, small p; p contains no unique DNA
- 13-15, 21-22, Y
What are the different types of chromosomal changes and how can each type be detected?
-
Numerical changes - Can be detected through:
- Traditional karyotyping
- FISH
- QF-PCR (Quantitative fluoresence PCR)
- NGS
-
Structural changes - Can be detected through:
- Traditional karyotyping
- FISH
What is meant by the term “Haploid”?
- One set of chromosomes (n=23) as in a normal gamete
What is meant by the term “Diploid”?
- Cell contains two sets of chromosomes (2n=46; normal in human)
What is meant by the term “Polyploid”?
- Any chromosome number which is an exact multiple of the haploid number e.g. 4n=92
What is meant by the term “Aneuploid”?
- Any chromosome number which is not an exact multiple of haploid number - due to extra or missing chromosome(s) e.g. 2n+1=47
What are the different types of numerical chromosomal abnormalities?
- Trisomy - Type of aneuploidy in which there are three instances of a particular chromosome, instead of the normal two
- Monosomy - Type of aneploidy in which there is only one instance of a particular chromosome
-
Mosaicism - When a person has 2 or more populations of cells with a different number of chromosomes
- E.g. person may have a population of haploid cells (46 chromosomes) and another population of cells with aneuploidy
Give a brief overview of Meiosis
- DNA is replicated so each chromosome has 2 sister chromatids
- Recombination occurs between homologous chromosomes
- Meiosis I: homologous chromosomes line up next to each other at the equator of the cell; get attached to the mitotic spindle and then get separated to opposite spindle poles
- Now each of the 2 cells has 23 pairs of chromosomes (diploid)
- Meiosis II: sister chromatids line up at the equator of the cell and get attached to the mitotic spindle and get separated to opposite spindle poles
- Now each of the 4 daughter cells only has 23 chromosomes (haploid)
Give a brief overview of Mitosis
- Prophase: Chromosomes condense, centrosomes move to opposite poles, mitotic spindle forms
- Prometaphase: Breakdown of nuclear envelope, chromosomes attach to mitotic spindle
- Metaphase: Centrosomes are at opposite poles, Homologous chromosomes line up one behind the other at the equator of the mitotic spindle and get attached to mitotic spindle
- Anaphase: Sister chromatids separated to opposite spindle poles
-
Telophase: chromosomes decondense, nuclear envelope reforms
- Now each of the 2 cells has 23 pairs of chromosomes (diploid)
What is it called when chromosomes/chromatids are pulled to opposite ends of the cell during anaphase?
- Disjunction
How does aneuploidy arise?
- Primary mechanism by which aneuploidy arises is non-disjunction - when homologous chromosomes DON’T separate from one another
What happens if non-disjunction occurs during meiosis I?
- If non-disjunction occur during meiosis I both copies of a pair of homologous chromosomes will end up in one daughter cell while the other daughter cell doesn’t get any
- Meiosis II will occur as normal so daughter gametes formed from cell that got both pairs of homologous chromosomes will end up with 2 copies of that particular chromosome (disomic)
- Daughter gametes formed from cell that didn’t get either pair of the homologous chromosomes don’t have any copies of that particular chromosomes (nullisomic)
What happens if non-disjunction occurs during meiosis II?
- If non-disjunction occurs during meiosis II then both sister chromatids of a particular chromosome end up in one daugther gamete while the other daughter gamete doesn’t get any
- This means one daughter gamete is disomic with respect to that chromosome while the other is nullisomic
Give some examples of the most common autosomal trisomies
- Trisomy 21
- Trisomy 18 (Edward’s syndrome)
- Trisomy 13 (Patau Syndrome)
What happens if non-disjunction occurs during mitosis (Mitotic non-disjunction)
- Initially results in one cell have 3 copies of a particular chromosome (trisomic) and one cell only having one copy of that same chromosome (monosomic)
- All monosomic cells are broken down
- Trisomic cells are maintained
- So end result is that you have some trisomic cells, some disomic cells as mitotic disjunction doesn’t occur in every cell and monosomic cells
- This is called mosaicism
What are the 2 mechanisms that result in mosaicism?
- Post-zygotic nondisjunction or mitotic non-disjunction: All diploid cells (2n) become a mixture of diploid (2n) and trisomic (2n+1) cells
-
Anaphase lag (trisomic rescue): Meiotic non-disfunction results in all cells being trisomic for a particular chromosome
- Every one of those trisomic cells goes through mitosis but during anaphase one of the 3 copies of that particular chromosome doesn’t connect to the mitotic spindle
- This results in a micronucleus forming around that chromosome which means it doesn’t end up in any of the daughter cells
- All daugther cells where anaphase lag occured are disomic (2n) and daughter cells where anaphase lag hasn’t lagged are still trisomic (2n+1)
What is the clinical relevance of mosaicism?
- Mosaic phenotype is thought to be less severe
- However, it’s difficult to assess:
- What proportion of cells will be trisomic/disomic
- What tissues/organs will be affected as a result of mosaicism
What are some examples of conditions caused by mosiacism in gametes?
- Turner’s syndrome - (46, XX / 45, XO)
- Klinefelter’s syndrome - (46, XY / 47, XXY)
How common are autosomal and gamete monosomies?
- Autosomal monosomies are very very rare
- Monosomies in gametes more common. e.g. Turner’s syndrome
What is the difference between full monosomy and partial monosomy?
- Full monosomy - Arises via meiotic non-disjunction
- Partial monosomy (microdeletion syndromes) - Far more common and mechanism different
What are all the different combinations of gametes that result in trisomic/monsomic zygote conditions
-
Nullisomic gametes
- O + X chr = XO = Turner’s (physically female)
- O + Y chr = lethal
-
Disomic gametes (XX)
- XX + X chr = XXX = Triple X syndrome
- XX + Y chr = XXY = Klinefelter’s (physically male)
-
Disomic gametes (XY)
- XY + X chr = XXY = Klinefelter’s
- XY + Y chr = XYY = XYY syndrome
How do you generate a karyotype from a patient to determine if there are any abnormalities?
- Entire process takes about 3 days
How can you analyse a developing foetus to see if it has any chromosomal abnormalities (prenatal diagnosis)?
- Chorionic Villus Sampling
- Amniocentesis
Give an overview of Chorionic Villus Sampling
- Transvaginal or transabdominal injection used to take sample of chorionic villus which is then analysed using karyotyping/FISH/QF-PCR
- Carried out at 11-14 weeks
- Miscarriage rate of 0.5% to 1%
- May also cause:
- Maternal contamination (getting maternal cells in sample)
- Transverse limb defects
Give an overview of Amniocentesis
- Injection used to take sample of amniotic fluid which is then analysed karyotyping/FISH/QF-PCR
- Carried out anytime after 16 weeks
- Miscarriage rate of 0.5-1%
What is G-banding?
- G-banding (Giemsa banding) involves staining chromosomes with giemsa stain
- Allows chromosomes to be viewed during metaphase
- Chromosomes are Lined-up based on:
- Size
- Banding
- Centromere position
- Dark bands = Heterochromatin
- Light bands = Euchromatin
Why do chromosomes viewed using G-banding have a badning pattern?
- Because chromosomes contain both euchromatin and heterochromatin and both stain differently when using the Giemsa stain
- Euchromatin = GC-rich; loosely packed; genes active
- Heterochromatin = AT-rich; tightly packed; genes inactive
Give a brief overview of FISH and explain how it works
- FISH = Fluorescent in situ hybridisation
- Uses cultured cells
- Looks at metaphase spread of chromosomes (same as karyotyping)
- Can detect abnormalities of up to 5-10Mb
- Design Fluorescent probe to a chromosomal region of interest
- Denature probe and target DNA
- Mix probe and target DNA together (hybridisation)
- Probe binds to the target DNA on the chromosome of interest
- Target fluoresces or lights up
What is QF-PCR and why is quicker to perform than FISH or karyotyping?
- QF-PCR (Quantitative fluorescence PCR) is a technique that uses fluorescent probes to amplify and then bind to specific microsatellites that are known to be on a specific chromosome
- QF-PCR is used to detect chromosomal abnormalities (normally trisomies) by quantifying the no. of a specific microsatellite
- E.G. If probes detect microsatellite X, known to be on chromosome 21, 3 times then you know it’s trisomy
- QF-PCR is quicker than FISH/Karyotyping because you don’t need to use cultured cells
How can you analyse a developing foetus to see if it has any chromosomal abnormalities, non-ivasively (Non-invasive diagnosis)?
- Cell-free foetal DNA (cffDNA) – DNA fragments in maternal plasma from 10 wks onwards
- They can be extracted and isolated from maternal blood and be tested to see if the foetus has any abnormalities
- For monogenic disorders you use PCR to amplify particular gene in foetal DNA which may cause particular disease before testing that gene
- For aneuploidies you use NGS to test foetal DNA
- SAFE TEST - Uses these principals to test for Trisomies 13, 18, 21 - these still do need to be confirmed using amniocentisis/CVS
