Study Unit 1 Flashcards
What materials are needed for karyotyping ?
Nucleated cells that can undergo cell division.
Example: 1. lymphocytes 2. Skin fibroblast 3. Bone marrow 4. Fetal cells in amniotic fluid
Karyotyping technique.
- Cultivate cells in tissue culture, stimulate to divide.
2 arrest cells in metaphase - Add hypotonic salt solution to swell cells for better spreading.
- Fix cells
- Drop cells onto microscope slides, releasing chromosomes.
- Stain with DNA binding dyes
- Visualise under light microscope
What substance stimulates mitosis ?
Phytohemgglutinin
Function of Colcemid
Stops mitosis in metaphase by inactivating spindle fiber formation.
Dark bands on a G-band represent?
AT rich area.
Highly condensed chromatin with little or no transcriptional activity will have a large portion of its histone protected from the trypsin and will therefore stain darkly following giemsa staining.
Light bands on a G-band represent ?
GC rich areas
Trypsin denatures euchromatic histones in DNA regions with higher transcriptional activity (loosely packed chromatin) resulting in light bands.
Pros G-banded karyotyping
- Whole genome analysis
- Overall impression
- Can use blindly, no prior knowledge required
- Can detect balance chromosome changes
- Can detect polyploidies and mosaics
Cons of G-banded karyotype.
- Low resolution (1-10Mb)
- Needs to be in metaphase thus cells need to be cultured.
- Labour intensive and specialised
- Time consuming, time delay to results (3-14 days)
Define Polyploidy and give examples
Definition: numerical change in a whole set of chromosomes.
Example: triploidy, there are 3 chromosomes in each set of chromosomes.
Define polysomy and give an example
Definition: only one set of chromosomes has an additional chromosome.
Example: trisomy 21, only chromosome 21 has 3 chromosomes.
What is fluorescence in situ hybridisation (FISH)
It’s a combination of karyotyping and hybridising techniques. It uses DNA probes labelled with fluorescent dyes to detect specific chromosomes/ chr regions using appropriate microscope.
FISH technique
- Fix chr on slides and treat to denature the dna to single strands.
- Prepare DNA probe
- Hybride fluorescently-labelled probes of interest to the DNA.
- Localize fluorescent signals against a background stain that binds to all the DNA sequences.
Different probes may be used in FISH.
- Gene-or locus-specific probes
- Repetitive DNA probes
- Whole chromosome probes
- fluorochromosomes.
Gene- or locus specific probes are used for
For presence, absence, location of particular gene.
Repetitive DNA probes used for
- Centromere repeats or telomere repeats
- To detect number of copies of particular chr
Whole chromosome probes are used for
- Chromosome painting
- Pool of fragments that covers entire chr/ chr arm
- Useful to detect chr rearrangements
What type of karyotyping Can evaluate complete karyote in single experiment or detect chr rearrangements in cancer cells, as well as different ploidy?
Spectral karyotyping (SKY)
Explain spectral karyotyping (SKY)
- 24 different chr-painting probes, one for each chr.
- Individual chr obtained by flow cytometry.
- Each chr labelled with specific combination of fluorescent dyes, to give unique fluore
- Signals analysed by computer, assigned a specific colour in order to generate photograph.
FISH pros
- Higher resolution
- Can use both inter- and metaphase cells
- Quick technique to detect abnormal chromosomes numbers.
- Many fluorochromes availed thus we can detect multiple probes at once/simultaneously.
FISH Cons
- Need prior information like the sequence of areas of interest
- Information gained is limited to design of probe
- Not general screening tool.
When studying the human genome what parts of the DNA were more focused on and what parts were less focused?
- Less focused on: The heterochromatin (tightly packed chromatin believed to be transcriptionally inactive) . Heterochromatin is believed to contain very few genes and non coding tandem repeats.
- More focused on: euchromatin( less tightly packed and transcriptionally active
Why do we still need to use karyotyping if there are more advanced methods to study chromosomes such as array CGH.
That is so because molecular genetic methods such as array CGH are not suited to detecting balanced chromosome rearrangements in which there is no net gain or loss of DNA. Inversions and balanced translocations would normally be invisible to these methods, but they can be detected by chromosome banding .
Chromosome FISH is used in what ways.
- Confirm regions of chromosome duplication or deletion.
- Screen for amplification of specific oncogenes.
- Used to detect recurring translocations that are often associated with cancer.
Single gene (monogenic) disorders
- Disorders determined by the alleles at a single locus.
- Characterised by specific patterns of transmission in families.
Recessive disorders
- Mostly due to loss-of-function mutation of a protein
- One functional allele expresses sufficient (50%) protein for normal physiological function.
Dominant disorders
- Manifest despite protein being expressed from normal allele.
- Can be pure dominance or incomplete dominance.
Explain pure dominance disorders .
Disorder is equally severe in AA and Aa (this is rarely the case for most dominant disorders)
Explain incomplete dominance disorders.
Disorder is more severe in AA than in Aa.
Examples of autosomal recessive inheritance.
Often errors of metabolism, defective enzymes/ receptors.
1. Cystic fibrosis
2. Oculocutaneous albinism
3. Phenylketonuria
4. Tay-sachs disease
5. Hurler syndrome
Why does consanguinity increase risk for autosomal recessive disease inheritance.
Blood relatives are more likely to share the same mutant allele, inherited from a common ancestor.
Characteristics of autosomal dominant inheritance.
- Phenotype occurs in every generation
- Each affected person has at least one affected parent.
- Affects males and females equally frequent and are equally likely to to transmit the trait.
Examples of autosomal dominant disorders.
- Marfan syndrome
- Ehlers Danlos syndrome
- Familial hyper cholesterolemia
Sometimes child is born with severe dominant disease, but family has no prior history. How is this possible.
This is due to a new mutation in the child that must have arose in either the maternal of paternal gamete.
Maintenance of defective allele in population depends on fitness of heterozygotes. Explain
- Can he/she reproduce
- If not, disorder can only manifest if affected person received defective allele from gametes of normal parents.
Explain X-linked inheritance
- Trait is determined by loci that occur on X chromosome.
- Females can be homozygous or heterozygotes
- No male -to-male transmission.
State some X-linked recessive disorders
- Haemophilia A and B
- Ichthyosis (certain types)
- SCID-X1
Female heterozygotes of X-linked disorders shows variable expression. Why?
For females carriers of X-linked disorders, X chr inactivated pattern will influence clinical expression. This depends on the fraction of cells in which normal allele is a active: mutant allele is active.
X-linked dominant inheritance
- Is regulated expressed in female heterozygote.
- Affected fathers will 100% pass of the gene to there daughters but will not pass the gene to there sons.
- Mothers offspring regardless of the gender have a 50% change of passing if hetero and 100% of passing if homo.
- Males often severe affects/ lethal before birth.
Examples of X-linked dominant inheritance disorders
- Incontinentia pigmenti
- Congenital hypertrichosis
- Rhett syndrome
Explain Y-linked inheritance
- Passed from father to son on the Y chromosome.
- No human disorder currently known to be transmitted in this way
- All sons of affected male will be affected none of his daughters will be affected.
Will female identical twins show variation when it comes to X linked recessive disorders
Yes, because of X chromosome inactivation is random and will happen randomly in each twin.
Why are Modes of inheritance seldom unambiguous.
- Cannot be completely certain by inspecting a single pedigree.
- If there is a Limited number of children, proportion of affected individuals is not reliable.
- For recessive conditions, carrier parents may by good fortune not have an affected child.
- Only when cloned copy of gene is available, can inheritance be defined with certainty.
What is mitochondrial inheritance
- Mitochondria and hence mtDNA supplied by ovum (not sperm) to zygote.
- Defective mitochondrial gene thus transmitted from mother to offspring
- Thus affected father cannot transmit disease
Heteroplasmy
Mitochondrial genome can include normal and mutant mtDNA because of the mitochondria distributed randomly to cells during mitosis thus expression of disorder phenotype depends on ratio of mutant mtDNA: normal mtDNA.
MtDNA disorders are characterised by?
- Reduced penetrants
- Variable expression
- Pleiotropy
Difference between X and Y chromosome
- X large—155Mb, gene rich
- Y small - 59Mb, gene poor
What is a pseudo-autosomal region
Homologous sequences on the X and Y chromosome
PSEUDO-AUTOSOMAL regions are required ?
- Required for X-Y pairing in male meiosis
- Important for correct segregation of chromosomes
- X and Y chromosomes can exchange sequences only here
Characterised PAR1 (found on Xp and Yp)
- Site of obligatory cross-over
- 5Kb from SRY gene
Characterised PAR2 (found on tips Xq and Yq)
Cross-over not necessary or sufficient for meiosis
Characterise the X-chromosome
- Gene- rich with many important genes.
- Majority of genes on X-chromosome not involved in determining gender.
- Encodes house-keeping and specialised functions
Characterise the Y chromosome
- Relative gene-poor
- Large portion is composed of heterochromatin which consists of repetitive, non-coding DNA
- Carries important SRY gene that determines maleness.
4.many Y-linked genes also show testis-specific expression that is important for normal spermatogenesis.
What Regions are deleted in azoospermia
- AZFa
- AZFb
- AZFc
Explain X chromosome inactivation
- In somatic cells of all females one X-chromosome is inactivated, only one is transcriptionally activate.
- This involves modification of chromatin structure: appears as heterochromatic Barr body in interphase cells .
Why does X-chromosome inactivation happen.
- It is a mechanism of dosage compensation to ensure that male and female somatic cells have equivalent quantities of proteins encoded from X-linked genes.
When does X-chromosome inactivation occur
X-chromosome inactivation initiated after embryo implants in uterus wall around 6 days post fertilisation, and once established, the silenced state in a cell is stably transmitted to all daughter cells.
Is X-chromosome inactivation passed on to the next generation.
X-chromosome inactivation pattern is erased between generations
Barr bodies
Inactive X-chromosome in a cell
Key players in X-chromosome inactivation.
- Xic (X inactivation centre)
- Distal RNA
- Epigenetic mechanisms
What role does Xic have in X-chromosome inactivation.
Minimal region on X-chromosome both necessary and require to trigger X-chromosome inactivation.
What role does Xist RNA have in X-chromosome inactivation.
- Essential for X-chromosome inactivation, by up-regulating prior to X-chromosome inactivation.
- 17 Kb IncRNA
- It is exclusively expressed from the future Xi
- Coats Xi over its entire length in cis.
What role does Epigenetic mechanisms have in X-chromosome inactivation.
- Repressive marks e.g methylated CpG islands, H3K9me3, H3K27me3, H4 deacetylation.
- Polycomb group complex
How many genes Escape from X-chromosome inactivation.
15%
What genes escape X-chromosome inactivation.
- Genes mostly located on Xp
- Genes with functional counterparts on Y-chromosome: have same dosage in males and females.
- Genes in PAR
Skewed X-chromosome inactivation causes disease, How?
Translocation between X-chromosome and autosome that disrupts a NB gene on X-chromosome.
In what circumstances might it be a good option to revert back to G-banding from FISH.
- When there is no clinical indication of a specific chromosomal abnormality. (General screening)
- If specific type of rearrangement or region/ sequence is not known.
- If a very large or balance rearrangement is suspected.
- To verify results obtained from other techniques.
Why study amniotic fluid instead of blood in karyotype.
Prenatal diagnosis
Why study chorionic villi instead of blood in karyotype.
High risk pregnancy, late gestation age (18-20 weeks)
Why study cord blood instead of blood in karyotype.
High-risk pregnancy, late gestation age (18-20 week)
Why study bone marrow instead of blood in karyotype.
- Diagnosis and classification of malignant haematological disorders. Prognosis, monitoring effects of treatment, monitoring patients in remission.
Why study skin instead of blood in karyotype.
- To study Mosaic congenital chromosomal abnormalities
2.new-born babies if blood aspiration fails
Why study solid tumours instead of blood in karyotyping.
Classification of malignant tumours associated with chromosomal abnormalities.
