Clinical Diagnostics

Question 1

What are some examples of chromosomal abnormalities

Answer 1

Aneuploidy due to non-disjunction
Translocations
Duplications/Insertions
Microdeletions

Question 2

What are the three cytogenetic studies

Answer 2

Karyotype
FISH
Array-CGH

Question 3

Karyotype Vs Array CGH for large chromosomal abnormalities

Answer 3

Array-CGH cannot reveal balanced translocations cos no gain or loss

Question 4

How does non-disjunction cause aneuploidy

Answer 4

Meiosis I - paired chromosomal fail to separate

Meiosis II - sister chromatids fail to separate

Question 5

What are the clinical features of Down Syndrome

Answer 5

ANS

Question 6

What are the clinical features of Patau’s and Edward’s syndrome

Answer 6

18 Patau’s - cleft lip, extra fingers, mid part of brain fused

18 Edward’s - clenched hand, rocker bottom feet

Question 7

What are translocations

Answer 7

Two different chromosomes sustain single break and incorrectly joined

Results in exchange of material between non-homologues chromosomes

Question 8

What are the types of translocation

Answer 8

Balanced - no deleterious phenotype unless it affects regulation of a gene

Unbalanced = too much or little of a chromosome

Reciprocal - when reciprocal parent gamete undergoes meiotic division, it cannot line up properly = exchange of material between non-homologous chromosomes

Robertsonian - acrocentric chromosomes involved, they may fuse
When having a baby this may lead to a loss or a trisomy

Question 9

What is DiGeorge Syndrome

Answer 9

22q11 microdeletion

Question 10

How is can DiGeorge Syndrome be diagnosed using clinical diagnostics

Answer 10

Can’t use FISH as it is too small of a deletion, and only if you’re certain of the diagnosis

Array-CGH used instead

Question 11

What is fragile X syndrome

Answer 11

Fragile X syndrome (FXS) is a genetic disorder characterized by mild-to-moderate intellectual disability.

Question 12

What kind of disorder is fragile X - (dominant, recessive etc.)

Answer 12

X-linked dominant affecting X chromosome at Q27.3 FMR1 coding for FMRP
Caused by expansion in 5’ UTR CAG repeat

Question 13

Why is it difficult to test for fragile X syndrome

Answer 13

Most tests require PCR but as it is a CAG repeat it is GC rich thus poorly sequenced (high Tm)

There may be artefacts, misalignment etc.

Question 14

How is fragile X syndrome tested

Answer 14

Historically- microscope
Now - southern blotting to size by length
Triplet PCR

Question 15

What is southern blotting

Answer 15

Separation based on length

Question 16

Describe the process of southern blotting

Answer 16

Probe hybridises near the repeat, with ECORI restriction sites on either side of the repeat

Restriction enzymes digest the DNA, and this is then run on agarose gel

Following this is the transfer to nitrocellulose membrane using alkaline buffer

DNA is hybridised, X ray film applied and then radiation is applied to develop the film

Question 17

What is triplet primed PCR

Answer 17

Used for CAG repeat expansions - method depends on the disorder tested

For fragile X
Forward (2)
Fluorescent probe binding upstream of the repeat
Repeat probe binding randomly to the repeat regions

Reverse - binds the downstream flanking region only, or that region AND some of the repeat regions

This results in amplification of the repeat at different lengths creating a ladder
The larger the region the wider the ladder due to more possible binding sites for the primers

Output in electropherogram
This uses capillary electrophoresis to separate by fragment size, and fluorescence analysis

Question 18

Describe the triplet primed PCR

Answer 18

Forward (2)
Fluorescent probe binding upstream of the repeat
Repeat probe binding randomly to the repeat regions

Reverse - binds the downstream flanking region only, or that region AND some of the repeat regions

This results in amplification of the repeat at different lengths creating a ladder
The larger the region the wider the ladder due to more possible binding sites for the primers

Uses capillary electrophoresis and fluorescence analysis visualised in an electropherogram

Question 19

What are the complications with triplet primed PCR

Answer 19

AGG interruption within CAG regions leads to decreased peak height

Question 20

What are the pros of triplet primed PCR + electropherogram

Answer 20

Can be applied to many repeat expansion disorders

Less laborious, no radioactivity and uses modern lab equipment

Little DNA requires - high sample throughput
ingle cell testing - pre implantation test of embryo

Question 21

What are the cons of triplet primed PCR + electropherogram

Answer 21

Methylation cannot be detected

Cannot accurately measure expansions larger than those in mutation zone

Problem between premutation and full mutation expansion differentiation

Question 22

Why cant WGS be used for fragile X syndrome

Answer 22

Due to PCR problems