9, 10, 11 Flashcards by Jacob Lewis

What stain do you add in G banding

Trypsin - Leishmann Stain

AT regions go dark, GC regions are light

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How long does G banding take

30 mins to 4 hours

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How long does FISH take

10 mins to 1 hour

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When do you denature DNA in FISH

In metaphase or interphase

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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

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What % of the human genome is CNV

12%

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High copy number of CCL31 causes

decreased susceptibility to HIV

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Low copy number variation of FGGR3B

increases susceptibility to inflammatory autoimmune diseases

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What does an MLPA do?

Can look at copy number variations in upto 50 genomic imbalances at a time

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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

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Is Microarry CGH genome wide

Yes
It shows genomic imbalances (copy number variations)
High detection rates and high resolution

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How much blood do you need for a microarry CGH

3ml in EDTA

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Who do we test with microarrays

those with learning disability and dysmorphic infacnts

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How big must the imbalance be to detect on microarray CGH

Over 150kb

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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

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What is QF-PCR

PCR amplification of short tandem repeats using fluorescent probes. Products visualised and quantified

the short tandem repeats are chromosome specific

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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

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What is qPCR used for?

Quantitive comparison to reference gene. Confirms small CNVs when FISH is unsuitable

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How much blood do we need for G banding

2-5ml, stimulate T lymphocytes, culture 2-3 days and then G band analysis performed

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Recurrence risk of Free Trisomy 21

95%

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Recurrence risk of Mosaicism Trisomy 21

If occurred post zygotic = minimal (47xx +21/46XX)

If occurred in meiosis = 1% (47X +21)

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Recurrence risk of 46XX der(21:21)

100% as 2 chromosome 21 stuck together

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Recurrence risk of 46XX der (14:21)

Male carrier = 2% as trisomy 21 sperm less viable

Female carrier = 12%

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What do we use to clarify imbalnces

Study These Flashcards

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

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

9, 10, 11 Flashcards

(58 cards)