GiM lectures 6,7,8 Flashcards Preview

P Intergrated Y2 > GiM lectures 6,7,8 > Flashcards

Flashcards in GiM lectures 6,7,8 Deck (65):
1

what is cytogenetics

study of chromosomes

2

The autosomes are which chromosomes?

1-22

3

X and Y are what type of chromsomes?

sex

4

What is conventional cytogenetics

metaphase chromosome analysis
- g banding

5

what is molecular cytogenetics

cytogenetic analysis at molecular resolution at all stages of cell cycle
- fish
- microarray CGH
- next generation sequencing
- MLPA
- QF - PCR
- qPCR

6

What are the phases of the cell cycle? (mitosis)
When are chromosomes most visible?

Most visible in metaphase

Interphase
Phrophase - line up
metaphase - spindle
anaphase - separate
telophase - cell split starts
cytokinesis - 2 diploid cells

7

G Banded metaphase shows what?

karyotype
30,000 genes
550 bands

8

What are the types of cytogenetic abnormality

Numerical
Structural

9

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

10

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

11

What causes errors at gametogenesis?
What is the result of errors in gametogenesis

increased maternal age
increased risk of aneuploidy

12

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

13

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

14

Triosmy 21 Clinical Features?
Frequency?
what % are miscarried?
Life expectancy?
Medical problems?

1/700
75% 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
Alzheimers
Hypothyroid
Diabetes, hearing loss, arthritis.

15

Edwards syndrome?
Frequency?
% miscarried
% survival to 1 year?
Clinical features?
Medical issues?

trisomy 18
1/6000 live births
95% miscarried
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
umbilical hernias
eye abnormalities - cataracts

16

Patau Syndrome?
Frequency?
% miscarried
Clinical Features

Trisomy 13
1/12,000 live births
95% miscarried

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

17

What is Turners?
What is Klinefelters?

Give frequency for all possible karyotypes

Turners 45,X 1/2,500

Klinefelters
47,XXY 1/1000
47 XXX 1/1000
47,XYY 1/1000

18

What are the clinical features of turners?

Reproductive = infertile
- Loss of ovarian function
- NO puberty

Lymphatic
- Webbed neck, swollen hands/feet

Other
short stature, coartation of aroma, IQ lower/normal

19

What are the clinical feature of klinefelter

Infertility
- testicular dysgenesis
- gynaecomastia

Growth
- very long arms and legs
IQ - normal

20

What errors can occur at fertilisation?

Polyploidy
Molar pregnancy - double paternal, no maternal input

21

What is Triploidy?
Karyotypes?
% of pregnancies?
% miscarried?
Frequency in live births?

Gain of whole set of chromosomes

69XXY, 69XYY, 69XXX
2% all pregnancies
99.9% miscarried
1/57000 live births

22

What are the origins of triploidy

Dingy = 2 maternal 1 paternal
Diplospermy = 1 maternal 2 from one sperm
Dispermy = 1 maternal, 2 sperm

23

What is caused by double maternal chromosomes?

Tiny placenta
Growth delay
head saving macrocephaly

Materal genome focus on foetus

24

What is caused by double paternal chromosomes?

Large placenta
some growth delay in foetus

Paternal DNA for placenta

25

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

26

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

27

What are the consequences of mosaicism?

Variable phenotype, non identical twin, recurrence risk

28

What are the possible types of balanced chromosomal rearrangement?

Balanced
1) translocation
- reciprocal
- Robersonian

2) Inversion
- pericentric
- paracentric

3) Insertion

29

What is reciprocal translocation
- how often does it occur
what is the phenotype risk?

2 chromosome break and exchange a section

1/500
5-10% phenotype risk

Has reproductive risk to offspring

30

What is a robertsonian translocation
Frequency?
Phenotype risk?

Whole arms fusion of ACROCENTRIC chromosomes
1/1000
no phenotype risk

has reproductive risk

31

What are inversions?
how often do they occur?
Phenotype risk?

2 breaks within a chromosome then rejoin

pericentric = around centromere
paracentric = swap within same limb

1/1000
5-10% phenotype

32

What are unbalanced rearrangements?
How often do they occur?

1/2000
copy number variation - gain or loss

commonest = deletions and duplication

affect several genes

33

What are the types of deletions?

1) Interstitial deletion - segment lost from one arm

2) Terminal deletion - end of arm lost

34

What are the types of duplications?

Direct - segment of chromosome copied twice
ab,ab

Indiverted - segment of chromosome is copied and inverted
ab,ba

35

What is caused by deletions and duplications?

abnormal phenotype due to abnormal gene dosage

36

What is a ring chromosome

where the chromosome breaks at either telomere and rejoins to form a circle

37

How is G banding carried out?

Cell culture
mitotic arrest
hypotonic
fixation
tyrpsin and leishman's stain
banding in AT and GC rich regions

38

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)

qPCR

39

What is the FiSH process

probe labelled with fluorochrome
Denature DNA
add target
hybridise
post hybridization washing
visualisation under UV light

40

What are the types of probes?

unique sequence - segment within chromosome
centromeric - centromere only
paints - for whole chromosome

41

When are centromeric probes useful

counting numbers of chromosomes

42

What are the applications of FISH

copy number imbalance
aneuploidy
confirmation of g banding
confirmation of array CGH
identifying specific abnormalities in cancer

43

What is a copy number variation

Consequences?

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

44

What is MLPA

Multiplex Ligation-dependent Probe Amplification
DNA-based
Multiplex PCR
Copy no. changes in up to 50 different genomic locations simultaneously
Alternative to FISH

45

What is Microarray CGH

Genome-wide screen

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)

detection rates

Replacing karyotyping as 1st line test

46

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

47

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

48

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

49

what is qPCR good for?

Quantitative comparison vs reference gene & normal control patient (amplify & quantify)

Confirming small CNVs

When FISH unsuitable

Primer design

50

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

51

What samples can be used for cytogenetics?

Which are appropriate for prenatal testing?

Blood

Amniotic fluid

Placenta

Other foetal tissue

Bone marrow

Tumour


Prenatal = amniotic fluid, chorionic villus

52

What leads to referrals for cytogenetic study?

Dysmorphic newborns, gender assignment, developmental problems, heart defect, reproductive problems, family studies

53

What is the recurrence risk of these robertsonian translocations
1) 46XXder(21:21)(q10:q10)
parent carrier

2) 46XXder(14:21)(q10;q10),+21
male carrier?
Female carrier?

1 = 100%

2 = male carrier = 2%
2= female carrier = 12%

54

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

55

What % recurrent miscarriages are caused by a genetic problem?

2-3% of sufferers of recurrent miscarriages have balanced chromosome changes

56

When is prenatal diagnosis carried out?

Amniocentesis 16weeks
chorionic villus biopsy 12weeks
NIPT 12 weeks

57

In a balanced translocation what can be formed?
give and example of a balanced translocation?

a quadrivalent will be formed

t(4;11) translocation

58

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

59

What are the indicators for prenatal diagnosis?

maternal age

serum screen risk

abnormal ultrasound scan (USS)

FH/previous chromosome abnormality

60

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

61

How is cytogenetic performed on chorionic villi?

1. Separate maternal from foetal tissue

2. QF-PCR

3. Culture cells (7-14 days) if QFPCR result abnormal

4. G-banded analysis

62

what are the advantages and disadvantages of array CGH and prenatal diagnosis

Replacement of cell culture if abnormal scan

Advantages
Increased resolution
Higher detection rate

Disadvantages
Ethical, eg small duplication & associated autism
Need parental follow up

63

What translocation can cause leukaemia

T(9;22) IN CML
"philadelphia chromosome

abl gene (9) fuses to bcr gene (22) - to create an abnormal protein

64

What forms of cytogenetics can be used on solid tumours?

Fresh - FISH/gBanding (1-20days)

Archived paraffin embedded tissues - FISH or genotyping

65

What does the presence of MYCN gene amplification in a neuroblastoma mean

poor prognosis
high dose chemo recommended