9, 10, 11 Flashcards
What stain do you add in G banding
Trypsin - Leishmann Stain
AT regions go dark, GC regions are light
How long does G banding take
30 mins to 4 hours
How long does FISH take
10 mins to 1 hour
When do you denature DNA in FISH
In metaphase or interphase
What are 3 types of FISH probe and what do you use them for
1) Unique sequence
2) Centromere - shows aneuploidies
3) Paint - shows translocations
What % of the human genome is CNV
12%
High copy number of CCL31 causes
decreased susceptibility to HIV
Low copy number variation of FGGR3B
increases susceptibility to inflammatory autoimmune diseases
What does an MLPA do?
Can look at copy number variations in upto 50 genomic imbalances at a time
Describe MLPA Process
50 different probes each with different stuffer sequence attached, add to DNA and hybridise, ligation reaction to join 2 halves of probes by thermostable ligase, PCR amplification using single piemr pair, analyse area under peaks
Is Microarry CGH genome wide
Yes
It shows genomic imbalances (copy number variations)
High detection rates and high resolution
How much blood do you need for a microarry CGH
3ml in EDTA
Who do we test with microarrays
those with learning disability and dysmorphic infacnts
How big must the imbalance be to detect on microarray CGH
Over 150kb
What are the disadvantages of micro arrays
Only measures dosage changes, not balanced rearrangements or mutations
doesn’t detect low levels mosaicism
Needs good quality DNA
non-pathogenic and uncertain pathogenic changes also detected
What is QF-PCR
PCR amplification of short tandem repeats using fluorescent probes. Products visualised and quantified
the short tandem repeats are chromosome specific
What does each box in a micro satellite tetra nucleotide repeat marker show?
Each box who’s a tetra nucleotide repeat. compare maternal and paternal values
What is qPCR used for?
Quantitive comparison to reference gene. Confirms small CNVs when FISH is unsuitable
How much blood do we need for G banding
2-5ml, stimulate T lymphocytes, culture 2-3 days and then G band analysis performed
Recurrence risk of Free Trisomy 21
95%
Recurrence risk of Mosaicism Trisomy 21
If occurred post zygotic = minimal (47xx +21/46XX)
If occurred in meiosis = 1% (47X +21)
Recurrence risk of 46XX der(21:21)
100% as 2 chromosome 21 stuck together
Recurrence risk of 46XX der (14:21)
Male carrier = 2% as trisomy 21 sperm less viable
Female carrier = 12%
What do we use to clarify imbalnces
FISH
Recurrent miscarriages often due to:
2-3% due to balanced chromosome change
what % of males infertile due to chromosome changes
10%
What chromosomes are prone to translocatinos
4 & 11
In meiotic pairing you get Pachytene Cross (quadrivalent forming) - also occurs in trisomy 21 roberstsonian translocation. Alternate controllers in the chain move to the same pole
What is Wolff Heschung condition
Loss of 4p chromosome
What is NIPT
Non-inasive Prenatal Testing. Take maternal blood, extract free circulating metal DNA. Assess aneuploidyies of 13,18,21 if high risk do invasive test
What indicates prenatal diagnosis
Increased maternal age, serum screen risk, abnormal USS, Family history/previous chromosome abnormality
Signs of downs syndrome
Serum test - low alphaFP, low estriol, high hCG
Nuchal thickness of 6mm
High maternal age
What causes 50% of spontaneous abortions
chromosome abnormalities
What type of abnormality links most cytogenetics and cacner
Translocations
What is MYGN gene amplification associated with
Neuroblastoma
Has poor prognosis, give high dose chemo
CML Translocation
between chr 9 abl gene and chr 22 bcr gene.
Creates bcr/abl fusion gene –> FISH cTan detect this
bcr/abl gives a Rh +ve interphase
What type of inheritance is Cockaynes syndrome
Autosomal recessive
Delays growth, causes premature ageing, degenerative condition, ataxia, photosensitivity and impaired development of the nervous system
Usually only live till about 7.
What does CK test for
Tests for muscular dystrophy
aCGH shows what?
Imbalances at chromosome level
Shoes copy number variations that can account for learning/behavioural/congenital phenotypes
Mutations are responsible for how much familial cancers?
16% in the under 35s
BRCA1/2 risk of breast and ovarian cancer
85% by 70 years for breast
55% for ovary BRCA1, and 25% for ovary for BRCA2
Treatments of BRCA
Remove ovaries (reduce risk by 85%), preventative mastectomy (reduces risk by 90%)
Specific treatments such as cisplatin and PARP inhibitors
Tamoxifen, diet and exercise also to reduce risk
What chromosome are the BRCA genes on
17
What is Treacher-Collins Syndrome
Problem in the migration of neural crest cells to the front of the face
Get small chin/narrow constricted airways
Can do prenatal screening (assess the need for a tracheostomy)
What week can you do a NGS of mothers blood
aka NIPT at about 10 weeks
What does leptin do?
decreases food intake, increase thermogenesis, increases physical activity and increase metabolic rate
THIS ALL CAUSES FAT CELL MASS TO DROP
What produces leptin
fat cells
What happens if you are leptin defecient
You can become obese –> use leptin replacement therapy
What is classical gene therapy
introduce functional genes/DNA to replace the mutated
What is newer gene technology
Repairs the mutated genes
What is Lebers Congenital Amaurosis
Rare inherited recessive eye disorder. Involved 22 genes. Causes 10-18% of congenital blindness
Why is Lebers Congenital Amaurosis a good target for gene therapy
as the eye is immune privileged (unlikely to mount an immune response), accessible for injection
Do sub retinal injections with aden-associated viruses containing human RPE65 and human RPE65 promoter
BUT need a structurally normal retina, only works with missions mutations and only a single gene with loss of function
Ambylopia may inhibit result so best to do in children
What mutation is seen in Lebers Congenital Amaurosis
Mutation in RPE65 (retinal pigment epihtelium)
It is involved in transduction recycling isomerase so it can capture light again
What vitamin is needed to capture light
Vitamin A
What are cytochrome P450 oxidases
Multilane family predominantly in the liver, responsible for the metabolic elimination of most drugs and the activation of pro-drugs to their active form
What cytochrome P450 metablises most drugs
CYP2D6
What % of caucasians are non-metabolites with no CYP2D6 activity and what % are ultra-rapid metabolites with multiple copies of CYP2D6
non-metabolites - 6/10%
ultra-metabolites - 7%
What is the rate limiting step in converting tamoxifen into its active form of endoxifen
CYP2D6
HENCE if poor CYP2D6 function then tamoxifen is not converted to its active form and is of no use
When do we give tamoxifen
give after a mastectomy to prevent prevent breast cancer
