Flashcards in GiM lectures 6,7,8 Deck (65):
what is cytogenetics
study of chromosomes
The autosomes are which chromosomes?
X and Y are what type of chromsomes?
What is conventional cytogenetics
metaphase chromosome analysis
- g banding
what is molecular cytogenetics
cytogenetic analysis at molecular resolution at all stages of cell cycle
- microarray CGH
- next generation sequencing
- QF - PCR
What are the phases of the cell cycle? (mitosis)
When are chromosomes most visible?
Most visible in metaphase
Phrophase - line up
metaphase - spindle
anaphase - separate
telophase - cell split starts
cytokinesis - 2 diploid cells
G Banded metaphase shows what?
What are the types of cytogenetic abnormality
How do cytogenetic abnormalities produce abnormal phentypes?
1) Dose effect (loss or gain)
2) disruption of gene
3) effect due to parental origin
4) position effect
- new chromosomal environment causes inappropiate gene function
5) unmasking recessive disorder
What are the numerical chromosome abnormalities
1) Aneuploidy - gain/loss trisomy/monosomy
2) Polyploidy - gain whole sets of chromosomes - triploidy, tetraploidy
3) mosaicism - diploidy and aneuploidy
diploidy - 2 copies of each chromosome
What causes errors at gametogenesis?
What is the result of errors in gametogenesis
increased maternal age
increased risk of aneuploidy
What are meiotic errors?
What percentage occur in meiosis 1 and meiosis 2?
Failure of chromosomes or chromatid separation
Meiosis 1 = 80-90% - chromosome non disjunction
Meiosis 2= 10-20%- chromatid non disjunction
What is the results of meiotic errors?
- Meiosis 1
- Meiosis 2
Meiosis 1 error = 2 disomic gametes, 2 nullisomic gametes
Meiosis 2 error = 1 disomic gamete, 1 nullisomic gamete, 2 normal gametes
Triosmy 21 Clinical Features?
what % are miscarried?
Head - upwards slanting eyes, small noes, low set ears, protruding tongue, flat face, brachycephalic, short neck
Learning disabilities IQ30-60
Single palmar crease, 5th finger clinodactyly, wide sandal gap
Males infertile (women fertile)
Life expectancy 55-68yo
Increased risk of leukaemia
Diabetes, hearing loss, arthritis.
% survival to 1 year?
1/6000 live births
10% survive to 1
Microcephaly, low set ears, micrognathia, cleft lip and palate, clenched hands, rocker bottom feet, low birth weight, short sternum, severe mental retardation
90% congenital heart disease
congenital kidney abnormalities
eye abnormalities - cataracts
1/12,000 live births
Small at birth, severe mental retardation, microcephaly, holoprosencephaly defect of brain, palpebral figures slanted, retinal dysplasia, microphthaia, cleft lip and palate, low set abnormal ears, polydactyly, heart defect, abnormal genitals
What is Turners?
What is Klinefelters?
Give frequency for all possible karyotypes
Turners 45,X 1/2,500
47 XXX 1/1000
What are the clinical features of turners?
Reproductive = infertile
- Loss of ovarian function
- NO puberty
- Webbed neck, swollen hands/feet
short stature, coartation of aroma, IQ lower/normal
What are the clinical feature of klinefelter
- testicular dysgenesis
- very long arms and legs
IQ - normal
What errors can occur at fertilisation?
Molar pregnancy - double paternal, no maternal input
What is Triploidy?
% of pregnancies?
Frequency in live births?
Gain of whole set of chromosomes
69XXY, 69XYY, 69XXX
2% all pregnancies
1/57000 live births
What are the origins of triploidy
Dingy = 2 maternal 1 paternal
Diplospermy = 1 maternal 2 from one sperm
Dispermy = 1 maternal, 2 sperm
What is caused by double maternal chromosomes?
head saving macrocephaly
Materal genome focus on foetus
What is caused by double paternal chromosomes?
some growth delay in foetus
Paternal DNA for placenta
what is a molar pregnancy?
Haploid sperm fertilises empty egg creating HAPLOID zygote.
Haploid zygote "doubles up" to create a diploid zygote.
DOUBLE paternal genome
conception without and embryo results in MASSIVE cystic placenta
What errors occur at early cleavage?
Where can errors occur?
What are the possibly karyotypes?
Mosaicism - mitotic non-disjunction
Errors can occur in second mitotic division
Monosomy, trisomy, normal disomy
What are the consequences of mosaicism?
Variable phenotype, non identical twin, recurrence risk
What are the possible types of balanced chromosomal rearrangement?
What is reciprocal translocation
- how often does it occur
what is the phenotype risk?
2 chromosome break and exchange a section
5-10% phenotype risk
Has reproductive risk to offspring
What is a robertsonian translocation
Whole arms fusion of ACROCENTRIC chromosomes
no phenotype risk
has reproductive risk
What are inversions?
how often do they occur?
2 breaks within a chromosome then rejoin
pericentric = around centromere
paracentric = swap within same limb
What are unbalanced rearrangements?
How often do they occur?
copy number variation - gain or loss
commonest = deletions and duplication
affect several genes
What are the types of deletions?
1) Interstitial deletion - segment lost from one arm
2) Terminal deletion - end of arm lost
What are the types of duplications?
Direct - segment of chromosome copied twice
Indiverted - segment of chromosome is copied and inverted
What is caused by deletions and duplications?
abnormal phenotype due to abnormal gene dosage
What is a ring chromosome
where the chromosome breaks at either telomere and rejoins to form a circle
How is G banding carried out?
tyrpsin and leishman's stain
banding in AT and GC rich regions
What are molecular cytogenetic techniques?
Fluorescent in situ hybridisation (FISH)
Multiplex ligation dependent probe amplification (MLPA)
Microarray comparative genomic hybridisation (array CGH)
Next generation sequencing
Quantitative fluorescent PCR (QF-PCR)
What is the FiSH process
probe labelled with fluorochrome
post hybridization washing
visualisation under UV light
What are the types of probes?
unique sequence - segment within chromosome
centromeric - centromere only
paints - for whole chromosome
When are centromeric probes useful
counting numbers of chromosomes
What are the applications of FISH
copy number imbalance
confirmation of g banding
confirmation of array CGH
identifying specific abnormalities in cancer
What is a copy number variation
DNA segment with a variable copy no compared with reference genome
Can give disease susceptibility OR resistance
Low copy no of FCGR3B = high susceptibility to inflammatory autoimmune disorders
High copy no CCL3L1 low HIV susceptibilty
What is MLPA
Multiplex Ligation-dependent Probe Amplification
Copy no. changes in up to 50 different genomic locations simultaneously
Alternative to FISH
What is Microarray CGH
Hybridise sample & control DNA to a microarray “chip” 1000s of DNA spots (oligonucleotides)
Genomic imbalances (copy number variants) at high resolution (10-10000x conventional cytogenetics)
Replacing karyotyping as 1st line test
What are the advantages of array CGH
Early diagnosis -1st line test, reduces need for other tests and avoids the “diagnostic odyssey”
High resolution = increased diagnostic hit rate
Greater accuracy of location/size of imbalances
Information on relevant genes
What are the disadvantages of array CGH
Dosage changes only – not balanced rearrangements or mutations
Low level mosaics not detected
Non-pathogenic & uncertain pathogenic changes detected
Needs good quality DNA
What is Quantitative fluorescent PCR (QF-PCR
PCR amplification of short tandem repeats (STRs) [chromosome-specific, repeated DNA sequences] using fluorescent primers
Products visualised & quantified as peak areas using an automated DNA sequencer
what is qPCR good for?
Quantitative comparison vs reference gene & normal control patient (amplify & quantify)
Confirming small CNVs
When FISH unsuitable
How does qPCR work?
Relative Quantitation (RQ) - compares difference in concentration between patient sample & normal control assessed by 2 different primer sets
RQ value is expressed as a ratio relative to 1 - a deletion has an expected value of 0.5 & a duplication an expected value of 1.5
What samples can be used for cytogenetics?
Which are appropriate for prenatal testing?
Other foetal tissue
Prenatal = amniotic fluid, chorionic villus
What leads to referrals for cytogenetic study?
Dysmorphic newborns, gender assignment, developmental problems, heart defect, reproductive problems, family studies
What is the recurrence risk of these robertsonian translocations
1 = 100%
2 = male carrier = 2%
2= female carrier = 12%
What is meiotic pairing
occurs in robertsonian translocation
- trivalent = 14;21 combined chromatic formed in robertsonian translocation undergoes pachytene with its sister 21 and 14 chromatids forming a 3 chromatid structure.
- a trivalent formation occur at PACHYTENE
(pachytene is where two sister chromatids separate from each other and cross over)
- Trivalent forms a chain during anaphase in meiosis 1
- centromeres move alternate poles
What % recurrent miscarriages are caused by a genetic problem?
2-3% of sufferers of recurrent miscarriages have balanced chromosome changes
When is prenatal diagnosis carried out?
chorionic villus biopsy 12weeks
NIPT 12 weeks
In a balanced translocation what can be formed?
give and example of a balanced translocation?
a quadrivalent will be formed
What are examples of NIPT (non invasive pre natal testing?)
What are the benefits and what does it test for?
Maternal blood sample
Extract circulating free fetal DNA
Assess aneuploidy of 13, 18, 21 (NGS)
Risk for aneuploidy – invasive test to confirm
Reduces no. of invasive tests
What are the indicators for prenatal diagnosis?
serum screen risk
abnormal ultrasound scan (USS)
FH/previous chromosome abnormality
How is cytogenetics performed on amniotic fluid?
1. Portion for DNA extraction (QF-PCR)
2. Separate cells from remaining fluid
3. Culture cells (7-14 days) if QFPCR result abnormal
4. G-banded analysis
How is cytogenetic performed on chorionic villi?
1. Separate maternal from foetal tissue
3. Culture cells (7-14 days) if QFPCR result abnormal
4. G-banded analysis
what are the advantages and disadvantages of array CGH and prenatal diagnosis
Replacement of cell culture if abnormal scan
Higher detection rate
Ethical, eg small duplication & associated autism
Need parental follow up
What translocation can cause leukaemia
T(9;22) IN CML
abl gene (9) fuses to bcr gene (22) - to create an abnormal protein
What forms of cytogenetics can be used on solid tumours?
Fresh - FISH/gBanding (1-20days)
Archived paraffin embedded tissues - FISH or genotyping