Cytogenetics (meg- finished) Flashcards
What are cytogenetics?
refers to the study of tissue, blood, bone marrow or culture cells in a lab, using banding or manipulating techniques to look for changes in the chromosomes, including broken, missing, rearranged or extra chromosomes
How are particular chromosomal disorders visualised to identify abnormalities?
2 main categories of analysis:
1. Microscopy based: Karyotype + FISH
2. DNA based: Array and PCR
What do chromosomal analyses test for?
Test for loss or gain of whole chromosomes, translocations, deletions (kbs to Mbs) or other rearrangements
Define: translocation
moving parts of a chromosome to other chromosomes
Name 2 clinical applications for chromosomal analysis
diagnosis of genetic syndromes
prenatal diagnosis
What is the difference between a male and female jumping jack ant in terms of their chromosomes?
Females have one pair of chromosomes = diploid
Males have only one chromosome = haploid
How many chromosomes does an adders tongue fern have?
631
What mammal has the highest number of chromosomes and how many do they have?
Plains rat- 56 pairs of chromosomes
What is this picture showing?
= female human karyogram = 23 pairs of chromosomes
How are human chromosomes numbered?
Organised based upon size apart from chromosome 21 and 22- mistake made thought 22 was longer than 21
During what stage of cell division can chromosomes be seen best?
Metaphase
What is a karyotype analysis?
= technique to visualise human chromosomes- can also be applied to other organisms
- Some techniques are combined with fluorescent DNA probes = FISH
What phase have chromosomes undergone before karyotype analysis?
Mitosis- S phase = DNA replication = 2 copies of each chromosome
What is the method used to view mitotic chromosomes called?
Giemsa method
Describe the method used to view mitotic chromosomes for karyotype analysis
- white blood cells must be stimulated to divide by adding phytohemagglutinin
- Cultured and colcemid added = cells accumulate at metaphase
- Add potassium chloride = cells swell and are fixed in methanol acetic acid
- Drop from height onto slide = causes cell to burst open and methanol acetic acid evaporates = leaving chromosomes
- Treated with trypsin to get rid of proteins
- Stained with Giemsa = karyotype
What does the giemsa method give?
reproducible banding patterns that are chromosome-specific
What is paris nomenclature?
= system where particular banding patterns are seen on particular chromosomes
What does banding patterns on chromosomes relate to?
banding patterns relate to how densely packed the structure of DNA is either being heterochromatin or euchromatin which defines whether there is a black or white region on chromosome
What is the difference between heterochromatin and euchromatin?
Hetero = tightly packed form of DNA = stained darker
eu = loosely packed form of DNA = stained lighter
What can banding patterns determine?
whether there has been translocation to other parts of a chromosome
How are chromosomes categorised?
Based on location of the centromere
Name + define the 3 categories of chromosome?
- metacentric = centromere almost at centre
- sub-metacentric = centromere near to one end of chromosome
- acrocentric = very close to end of chromosome with small satellite regions
What is a satellite region?
= segment of a chromosome that is separated from the rest of the chromosome by secondary constrictions
What is another phrase for the centromere?
primary constriction
How are human chromosomes labelled?
Based on the length of their arms
What letters represent short and long arms of human chromosomes?
- Short arms = p
- long arms = q
Which human chromosomes have satellites?
associated with the short arm of an acrocentric chromosome:
-13
-14
-15
-21
-22
What do the acrocentric chromosomes contain and what can this lead to?
rDNA and repetitive sequences = can lead to lots of genetic outcomes
What is rDNA?
= ribosomal DNA = DNA important in the generation of RNA
Can chromosomal translocations be identified by karyotyping?
Yes
What is this picture showing?
chromosomal translocation e.g.
- Break that occurs on chromosome 1 on q arm and break that occurs on chromosome 22 on q arm
- These can recombine = end of chromosome 22 being translocated to end of chromosome 1- so chromosome 1 is shortened
- End of chromosome 1 translocated to end of chromosome 22- chromosome 22 is lengthened
do chromosomal translocations always lead to a disease outcome and why is this?
may not be producing any kind of disease outcome:
- Often balanced so no difference in DNA pre translocation and post translocation- not always pathogenic
- might not be a problem associated with mitosis so not problem with individual
Describe how chromosomal translocations can cause problems
- If the breakpoint within an important gene / promoter (= region of DNA that facilitates the transcription of a particular gene) can cause problems- may be associated with specific cancers
- Often causes problems in meiosis as one copy of genome from mum and one copy from dad – if one copy is altered then cannot pair with homologous chromosome
Name 5 other types of chromosomal translocations and where have all of these been observed?
Lots of these translocations are observed in cancers
- terminal deletion
- interstitial deletion
- Inversions
- Duplications
- Ring chromosome
What is a terminal deletion?
= break in chromosome resulting in loss of DNA
What is a interstitial deletion?
breaks on same chromosome at different loci- this whole region could be removed = loss of genetic info
What are inversions?
break at different loci on same chromosome and it can switch around- may not be problematic
What are duplications?
= regions that have been copied
what is a ring chromosome?
= breaks occur on particular chromosomes and telomere is lost
What is a telomere?
region of repetitive DNA sequences at the end of a chromosome
Name + define an extreme case of a chromosome translocation
Chromothripsis = chromosome shattering
What does chromothripsis look like?
Looks like catastrophic fragmentation and rearrangement of one chromosome, usually with loss of material
What is this picture showing?
= chromothripsis
Original chromosome sequence, then catastrophic event occurs = complete shattering of DNA
= rearrangement of DNA from lots of different chromosomes + loss of DNA
When can chromothripsis be advantageous?
e.g. rearrangement of chromosome 2 = cure of WHIM syndrome
WHIM syndrome associated with CXCR4 gene which leads to disease outcome but on very rare occasions chromothripsis can occur = loss of CXCR4 gene = no WHIM syndrome
Can chromothripsis occur in normal cells?
Yes
What is a karyogram?
Human chromosomes arranged in order
What is this picture showing?
karyogram
What are advantages and disadvantages of karyotyping?
Homologous chromosomes can be identified by their size and banding patterns- can be used for:
+ Large changes, easily observable
+ Advantageous if you don’t know what you’re looking for
+ Quick and easy to do
- But can miss changes smaller than around 5Mb
What can be used instead of karyotyping when looking for smaller changes?
Fluorescent in-situ hybridisation (FISH) analysis
Describe the process of FISH
- Get DNA from region of interest- have to know what we’re looking for unlike karyotyping
- Clone this DNA
- Fluorescently label particular region of DNA
- Make preparation of chromosomes and fix on slide
- Hybridise DNA
- If region of interest exists it will bind to their DNA, which is detected as a fluorescent signal on their chromosome
What is this picture showing?
= Example of FISH
- 2 different loci- one in red and one in green
= individual has lost red locus
Important to have control probes (= chromosome with red + green loci)
What is array based genomic hybridisation?
= looking and comparing amount of particular genomic locus throughout genome compared to a control
When is array based genomic hybridisation useless?
useless if translocation occurs where it is balanced (no gain or loss
Name 3 DNA based methods
Array-CGH
SNP array hybridisation
STR analysis
What is array-CGH and what is it used for?
= Comparative genomic hybridisation- array
Used to identify gain or loss of chromosomal regions not visualised by karyotyping- smaller regions, usually about 1.6Mb
What does array-CGH not detect?
balanced translocations
Describe the process of array-CGH
- Cells from normal control + cells from patient- don’t need to know particular region
- Extract DNA from normal + patient cells
- These are labelled with 2 different fluorescent labels (= fluorochromes)
- Mix in equal quantities
- Hybridise them against microarray of clones across entire genome
- = read out of fluorescent levels for 2 different channels
What are the results suggesting in this picture?
RESULTS =
Regions can be identified where there are equal amounts of both types of DNA, then yellow is seen
Both in regions where DNA has been lost from patient = dots of red signal- so patient is missing part of DNA at that region
What is this graph suggesting (array CGH)?
chromosomes along bottom
= analysing signal from control compared to patient probe
= trace around 0 which indicates that they are equal
Apart from chromosome 21 where there is a 50% increase in signal in patient = 3 copies of chromosome 21
What is the difference between array CGH and SNP array hybridisation?
Array CGH allows for entire chromosome investigation but dependent on probes using as this defines how big or small a region is
- but development of SNP array hybridisation has allowed for investigation of specific loci where we know a particular mutation leads to a specific disease outcome
Describe the process of SNP array hybridisation
- Patient sample
- Sometimes undergoes amplification by PCR but lots of different probes can be put in to amplify entire genome or regions of interest
- Fragmentation = shear genome in lots of short parts
- These parts are fluorescently labelled
- Microarrays that are bound to particular glass slide that has specific SNP relating to a disease outcome
- Wash fragmented + labelled DNA over specific glass slide- if it hybridises and binds = fluorescent signal
- = regions can be identified with SNPs which might lead to disease outcomes
- Image them on high resolution fast array imager that detects fluorescent outcome for particular SNP
What is amplification?
the production of multiple copies of a sequence of DNA
What is an SNP?
Single nucleotide polymorphism
What is a DNA probe?
A single stranded and labelled piece of DNA that can hybridise with its complementary sequence in a sample genome
What is short tandem repeat assay?
Regions of genome where different individuals have different number of copies of particular region (=STRs) can be compared if with a satellite region in normal individual there might be different number of these STRs
What is an STR?
= short tandem repeat
= microsatellite with repeat units that are 2 to 7 base pairs in length
Describe the method of an STR assay for down syndrome
If looking to see if increased number of chromosome 21- can find satellite region on chromosome 21 and can PCR across that region to get a particular size based upon number of repeats they have at that region:
Describe the results of this STR assay
- For normal foetus = 2 peaks- indicating that they have 2 copies of chromosome 21 with different repeats at different regions
- Foetus with down syndrome = 3 peaks- have another satellite region from an extra chromosome
- Also foetus with down syndrome = 2 chromosomes with identical number of satellite repeats and 1 chromosome with different number of satellite repeats- so 2 peaks seen but one is amplified from double amount of DNA so is twice as high
Name an origin of chromsomal disorders
= many medically important chromosome changes arise in meiosis
Describe the process of meiosis I
- Reductional division in order to become a diploid individual otherwise each generation will have double amount of chromosomes
- Segregate homologous chromosomes correctly
- red + blue homolog – need to ensure each gamete has one copy of each pair of homologous chromosomes but these are not attached, so in order to attach these double strand breaks are introduced (= DNA damage) which repair as crossovers which link the homologous chromosomes enabling accurate segregation as well as genetic diversity
Describe the process of meiosis II
Meiosis II
Undergo similar division to mitosis = segregation of sister chromatids
Outcome = produces gametes that are sexually dimorphic
What is recombination and when does it occur?
= formation of double strand breaks and crossovers during meiosis I which enables accurate chromosome segregation in meiosis
Where does recombination occur?
at structures called chiasmata
why is recombination required?
correct separation of homologous chromosomes at anaphase
e.g. grasshopper chromosomes
What is this picture showing?
= Grasshopper chromosomes
Chiasmata = visual representation of crossover events
Part of blue chromosome becomes red = attaches 2 homologous chromosomes enabling accurate segregation
What does recombination lead to?
genetic diversity
recombination in meiosis
Describe what this picture is showing
- A and B represent DNA variants (polymorphisms)
- The closer together A and B are, the less likely there is a cross-over between them (left picture)
random assortment of chromosomes into gametes = different combinations of gene variants (right picture)
What can be used to track the inheritance of these polymorphisms?
In linkage analysis, DNA markers are used to track inheritance of polymorphisms
Does mammalian meiosis differe between males and females?
Yes
Describe the process of meiosis in females
In females meiosis is known as oogenesis- start off with diploid cell
1st stage happens in womb- then arrest after recombination at a point called dictyate arrest- instead of dividing and producing 2 equal cells, they segregate and extrude a polar body
Sits there until its ovulated and if fertilised then 2nd meiotic division occurs- where other polar bodies are extruded
= oocyte
Name 4 key points of female meiosis in humans
- Meiosis starts in the female fetal germline
- Fetal meiosis arrests after recombination – it pauses = dictyate arrest
- Meiosis resumes at ovulation, then stops again
- Meiosis only finally completes after fertilisation
Describe some differences between meiosis in females vs males
Meiosis starts in the female fetal germline – in males spermatogenesis isn’t initiated until post puberty
In males- sperm cell can go through all of spermatogenesis from an initial germ cell through to fully formed spermatozoa in a few weeks
Oocytes completed between 20-40 years
Define: germ cell
precursor diploid cell that develop into gametes
female meiosis
Describe what this picture is showing
- Primordial germ cells enter meiosis after 11-12 weeks of gestation
- Enters prophase and complete recombination - Enters dictyate arrest
- At ovulation they resume meiosis and completes meiosis I = segregate 1st polar body
- Oocyte arrests again in meiosis II
- Fertilisation triggers the completion of meiosis II
Define: aneuploidy
where an organism or cell has only one or a few chromosomes added or missing
Define: trisomy
= extra copy of chromosome = 3 copies
Outline 3 examples of trisomy
- Down syndrome = trisomy 21 = 3 copies of chromosome 21 (most common) or extra copies of part of chromosome 21
- Patau syndrome = trisomy 13- most babies dies within 6 months
- Edwards syndrome = trisomy 18- 50% of babies die within 1st month
Where does most aneuploidy occur?
Human embryos have high rates of aneuploidy- most aneuploidy occurs from oocytes
What % of sperm and oocytes have aneuploidy?
1-2% of sperm have aneuploidy
20% of total oocytes in female have aneuploidy
What is the largest risk factor for trisomies?
Maternal age
Why is aneuploidy a major cause of developmental and cognitive disabilities?
Due to the gene dosage effect- for normal function 2 chromosome copies are required
Are there syndromes associated with abnormal sex chromosome numbers?
Yes but these are much less deleterious than wrong number of autosomes
Why are abnormal sex chromosome numbers less deleterious?
X inactivation reduces the effect of extra X chromosome = one X chromosome is normal dosage for humans as women have 2 they have to shut down one of their 2 X chromosomes
= increased copy numbers of X chromosomes can be dealt with as X inactivation dampens down extra X chromosome
What syndromes can arise due to abnormal sex chromosome number?
- Klinefelter syndrome (47,XXY)- defined as male due to presence of Y chromosome = infertile, tall with female distribution of body fat
- Turner syndrome (45, X) = females with short stature, infertility, normal intelligence, increased risk of organ abnormalities
- Trisomy X syndrome (47, XXX) = affects females and is mostly undiagnosed due to X inactivation
- XYY syndrome = males, tall, other features uncertain = increased copy of Y with no X inactivation
How do increased copies of chromosomes arise?
Non disjunction of meiosis I and II
Describe how non-disjunction of meiosis I can lead to increased copies of chromosomes
Non disjunction of meiosis I =
- meiosis I = homologous chromosomes not segregated correctly = one empty cell + cell with double amount of DNA
- meiosis II = 2 empty gametes + 2 gametes with double amount of DNA
FERTILISATION by normal gamete = 4 potential zygotes = 2 situations of trisomy + 2 situations of monosomy (one copy of chromosome)
Describe how non-disjunction of meiosis II can lead to increased copies of chromosomes
Non dysjunction of meiosis II =
- Meiosis I = divide correctly
- Meiosis II = one cell divides correctly = 2 normal gametes + one cell divides incorrectly = one empty gamete + gamete with double amount of DNA
FERTLISATION by normal gametes = 4 potential zygotes = 1 case of trisomy + 1 case monosomy + 2 normal zygotes
What % of trisomy 21 cases are due to non-dysjunction in 1st meiotic division in the mother?
70%
When is clinical cytogenetic analysis used?
- often used when a child is suspected of having a genetic syndrome (not always typical)- Often associated with changes in chromosome number or large deletions
- Can also be used in prenatal testing especially if increased maternal age and for preimplantation genetic diagnosis
Describe the down syndrome phenotype aspects present in all patients
Aspects in all patients =
- Cognitive impairment
- Characteristic facial appearance
Describe the down syndrome phenotype aspects present in many but not all patients
Present in many but not all individuals:
- Congenital heart defect
- Acute megakaryocytic leukaemia
What do the differences of the down syndrome phenotype depend on?
Differences depend on whether it is translocation 21 or full increase copy number of chromosome 21
What disease is down syndrome associated with and what proportion of patients will be affected?
Association with Alzheimer’s disease
- 10% age 40-49
- 100% at age 70 years
Why is there an association with alzheimers disease and down syndrome?
This is because of the extra copy of gene for amyloid precursor protein that causes Alzheimer’s
What methods can be used to diagnose down syndrome?
Traditional karyotype
Interphase FISH
Array analysis
Short tandem (=microsatellite) repeat typing
What is an advantage for karyotyping to diagnose down syndrome?
+ distinguishes translocation Down’s = part of or whole chromosome 21 that has fused onto another chromosome as long as it is greater than 5 mega bases, it can be detected
What are microdeletion syndromes?
microdeletion between 1-5MB = 1-5 million bases- number of distinct clinical syndromes are caused by chromosomal microdeletions
What do microdeletion syndromes cause?
haploinsufficiency
Define: haploinsufficiency
A single copy of a gene is insufficient to maintain normal function
What methods can be used to detect microdeletion syndromes?
Not detected on karyotype, but detected by molecular techniques if particular region is known e.g. FISH, microarray
What are contiguous gene deletion syndromes and how do they arise?
= piece of DNA where multiple genes next to each other have been deleted
- Arise due to unequal cross-over events in meiosis
Name 4 examples of microdeletion syndromes
- VCFS = velocardiofacial syndrome
- WBS = williams-Beuren syndrome
- Angelman syndrome
- Prader-Willi syndrome
Describe a VCFS case study (Gillian) and what was suspected by the geneticist
Gillian = 2nd child of healthy parents- after birth, she was diagnosed with heart murmur (ventricular septal defect = hole in heart)
- 3 years- all developmental milestones were slower than normal
- Referred to geneticist who requested a chromosome test- karyogram appeared normal but deletion is between 3-5Mb so can’t be detected via karyogram
- Geneticist suspected a 22q11 deletion involved in VCFS due to combo of clinical symptoms
- FISH analysis carried out
What were the results for the FISH analysis of the VCFS case study?
Results from FISH =
Green = control probe for chromosome 22 showed 2 parts of signal
Red = patient probe only one region was detected suggested a deletion
= VCFS as disease outcome
What is WBS how many births does it effect?
= contiguous gene disorder- 1 in 15000-20000 births
What is WBS caused by?
Caused by 1.4Mb microdeletion on chromosome 7q (= long arm of chromosome 7)
What are the symptoms of WBS?
Symptoms = supravalvular aortic stenosis (SVAS) = pinching of valve restricting blood flow, hypercalcaemia = increase in calcium, mild learning disability, distinctive facial features, distinctive behavioural abnormalities
How was the cause of WBS discovered?
Family presented with SVAS but no other WBS symptoms:
- Cytogenetics showed that all affected individuals carried a balanced translocation between chromosome 6 at p21.1 and chromosome 7 at q11.23 disrupting the elastin gene on chromosome 7q11
Found that individuals with SVAS but not WBS have mutations on elastin gene
- Haploinsufficiency of elastin results in SVA so suggested that genes next to elastin gene are implicated in WBS- the deletion is about 1.4Mb + many genes are deleted
= Haploinsufficiency of one or more of these additional genes is responsible for other WBS characteristics
Describe the case study of Elizabeth (daughter) and Ellen (mother) + results of karyotyping
Elizabeth has undiagnosed syndrome
Ellen has history of miscarriage
Results from karyogram
- Ellens chromosomes = chromosomes 1 and 22 have exchanged material = balanced translocation
- Elizabeths chromosomes = inherited Ellens normal chromosome 1 + translocated chromosome 22 = had 3 copies of part of chromosome 1 and only one copy of chromosome 22
Describe what the results of the array CGH for ellen and elizabeth shows
Region of array suggests increased copy number of chromosome 1
Supporting karyogram
so ellen = constitutional carrier of a balanced translocation suggested to have been present in one of the maternal grandparents
What does the results of ellen and elizabeths case mean for the family?
Substantial risk of problems in future pregnancies
What are the advantages and disadvantages for SNP array hybridisation?
+ Enable detection of SNPs within patient sample
- Only detecting specific SNPs, usually associated with disease
What are the advantages and disadvantages for array CGH?
+ enable detection of duplications or deletions not visually detectable
- will miss balanced mutations
What are the advantages and disadvantages for STR analysis?
+ Comparing specific genetic markers
- Shorter sequences may amplify preferentially, sometimes providing confused results
What are the advantages and disadvantages for karyotyping?
+ Will see duplications and chromosomal rearrangements
- Will miss small changes- less than approx 5Mb
What are the advantages and disadvantages for FISH
+ Can detect small changes
- Have to know what you are looking for: specific probes and specific analyses
