structural chromosomal abnormalities

Question 1

describe structural abnormalities

Answer 1

translocations= reciprocal, robertsonian

inversion

deletion

duplication

rings

isochromsomes

Question 2

describe translocation

Answer 2

exchange of two segments between non-homologous chromosomes

reciprocal or robertsonian

Question 3

non homologous end joining

Answer 3

No net gain or loss of genetic material
Involve any chromosome and any size fragment
Common: 1 in 930 people

Plus copy of normal 1 and normal 22
Carrier of balanced translocation

Question 4

results of unbalanced reciprocal translocation

Answer 4

Many lead to miscarriage (hence why a woman with a high number of unexplained miscarriages should be screened for a balanced translocation)

Learning difficulties, physical disabilities

Tend to be specific to each individual so exact risks and clinical features vary

Question 5

robertsonian translocation

Answer 5

Two acrocentric chromosomes join near centromere with the loss of p arms
Balanced carrier has 45 chromosomes
If 46 chromosomes present including Robertsonian then must be unbalanced
p arms encode rRNA (multiple copies so not deleterious to lose some)
Robertsonian translocations 13;14 and 14;21 relatively common. 21;21 translocation leads to 100% risk of Down syndrome in fetus

Question 6

outcomes of translocations

Answer 6

Very difficult to predict
Only have approximate probability of producing possible gametes
Some unbalanced outcomes may lead to spontaneous abortion of conceptus so early that not seen as problem
Some unbalanced outcomes may lead to miscarriage later on and present clinically
Some may result in live-born baby with various problems

Question 7

deletions

Answer 7

1:7000 live births
Deletion may be terminal or interstitial
Causes a region of monosomy
Haploinsufficiency of some genes
Contiguous gene syndrome (multiple, unrelated clinical features)
Phenotype is specific for size and place on deletion
Gross deletions seen on metaphase spread on G-banded karyotype

Question 8

micro deletions

Answer 8

Many patients had no abnormality visible on metaphase spread
High resolution banding, FISH and now CGH showed ‘micro’ deletions
Only a few genes may be lost or gained
Velocardiofacial (DiGeorge), 22q11
Wolf-Hirschhorn, 4p16
Williams, 7q11
Smith-Magenis, 17p11

Question 9

source of sample

Answer 9

Prenatal
Amniocentesis
Chorionic villus sampling
Cell-free fetal DNA
Postnatal
Blood
Saliva

Question 10

chromosome staining

Answer 10

Most common = G-banding
G = Giemsa
Why bands?
Chromatin
2 different sorts: euchromatin & heterochromatin
Euchromatin = GC-rich; loosely packed; genes active
Heterochromatin = AT-rich; tightly packed; genes inactive
Stain differently

Question 11

summary of G banding

Answer 11

How does karyotype of patient differ from expected?
Uses a chemical stain
Uses metaphase chromosomes
Takes several days at least
Looks for aneuploidies, translocations & very large deletions

Question 12

FISH

Answer 12

Fluorescent in situ hybridisation
Hybridisation = single stranded nucleic acid strand binds to a new single stranded nucleic acid strand (DNA/DNA or DNA/RNA)
Cultured cells, metaphase spread
Fluorescent probe
Denature probe and target DNA
Mix probe and target DNA
Probe binds to target

Question 13

what is a probe

Answer 13

single stranded DNA (or RNA) molecule
Typically 20 – 1000 bases in length
Labelled with a fluorescent or luminescent molecule (less commonly a radioactive isotope)
In some techniques thousands or millions of probes are used simultaneously

Question 14

summary of FISH

Answer 14

How does karyotype of patient differ from expected?
Uses fluorescent probes for SPECIFIC parts of genome
Uses metaphase chromosomes
Takes several days at least
Looks for aneuploidies, translocations & large deletions
Traditional FISH

Question 15

array CGH:

Answer 15

Array comparative genomic hybridisation

For detection of sub-microscopic chromosomal abnormalities

Patient DNA labelled Green

Control DNA labelled Red

Question 16

summary of aCGH

Answer 16

How many copies of a particular genomic region does the patient have?
Uses fluorescent probes to differentiate between patient and control
Uses extracted DNA
Looks for microdeletions and microduplications

Question 17

QF-PCR

Answer 17

Quantitative fluorescence polymerase chain reaction
Trisomies 13, 18 and 21
Uses microsatellites

Question 18

micro satellites

Answer 18

Short repeated sequences
Number of repeats varies between individuals
Total length of microsatellite sequence varies between individuals

Question 19

detecting microsatellitles

Answer 19

Isolate DNA from individual
Design primers specific to flanking sequences
PCR amplification
Gel electrophoresis

Question 20

detecting micro satellites 2

Answer 20

PCR amplification of microsatellite region
Genotype size of fragments on gel-based system
Homozygotes = single product of specific size
Heterozygotes = two different sized products

Question 21

PCR

Answer 21

Exponential amplification of a DNA fragment of known sequence
Components of the PCR reaction:
Template – DNA to amplify
Primers – Short pieces of ssDNA (15-30bp)
Polymerase – thermostable enzyme (Taq)
Nucleotides – single base mixture (dNTPs)
Buffer – To maintain pH
MgCl2 – Essential for polymerase activity

Question 22

PCR 2

Answer 22

PCR consists of incubating at three different temperatures

This results in three different processes happening:
Denaturation
Annealing
Extension

Question 23

PCR exponential amplification

Answer 23

The PCR quickly reaches an exponential amplification phase…but remember it doesn’t go on forever!

The components of the PCR reaction are not unlimited so at some point the reaction will slow down and stop

Question 24

QF-PCR

Answer 24

Perform PCR using primers for microsatellite known to be on chromosome 21 (if testing for Down’s)
Should be two copies of microsatellite (one from mother, one from father, like any other autosomal locus, gene, whatever)
If homozygous, there will be a single peak of high signal
If heterozygous, there will be two peaks of similar, lower signal

Question 25

summary of QF-PCR

Answer 25

How many copies of a chromosome does the patient have?
Uses fluorescent probes for SPECIFIC microsatellite markers on SPECIFIC chromosomes
Uses extracted DNA
Quick (~48hrs)
Looks for aneuploidies
Need to know what you’re looking for

Question 26

non-invasive pre-natal testing and NGS

Answer 26

Cell free fetal DNA
Maternal blood sample
Trisomy testing
Next-generation sequencing
“High chance” indicator for invasive test

Question 27

summary of NIPT and NGS

Answer 27

Uses extracted DNA from mother
Looks for aneuploidies
Utilises next generation sequencing
Screening not diagnostic