Molecular aspects of Huntington's and triplet repeat conditions

Question 1

What are triplet repeats

Answer 1

repetitive sequences in genome (they are common in genome)

e.g. CAGCAGCAGCAG

Question 2

What does a longer repeat mean in neuromuscular and neurodegenerative disorders

Answer 2

Longer repeats=more severe disease phenotype

Question 3

What type of disease is Huntington’s disease

What gene is it to do with

Answer 3

Autosomal dominant

Expansion of the CAG trinucleotide repeat in exon 1 of the huntingtin (HTT) gene

Question 4

What is a normal CAG allele repeat size

Answer 4

<27

Question 5

Myotonic Dystrophy type 1 symptoms

Answer 5

Myotonia (difficulty relaxing clenched hands or a hand shake)

Muscular dystrophy
Cataracts

Testicular atrophy

frontal balding

Cardiac conduction defects

Question 6

Cause of Myotinic dystrophy type 1

Answer 6

expansion of a CTG trinucleotide repeat in the 3’UTR of the DMPK gene

Question 7

What happens to CTG repeats and what does this lead to

Answer 7

transcribed into mRNA, but not translated into protein

-Large mRNA molecules accumulate in the nucleus. These acquire a new toxic function by trapping essential cellular RNA-binding proteins

Question 8

Normal range of repeat size in Myotonic dystrophy type 1

Answer 8

5-35

Question 9

difference between mechanism of disease of Huntington’s and Myotonic dystrophy type 1

Answer 9

Huntingtons: CAG expansion in exon 1 of HTT gene (coding region)

Myotonic dystrophy:
CTG expansion in 3’UTR of DMPK (non-coding regions)

Question 10

Why can triplet repeats expand

Answer 10

Due to errors in DNA replication and repair (this accounts for normal variation in repeat length)

Question 11

When a triplet repeat reaches beyond a certain size, what happens

Answer 11

It becomes more unstable and more likely to expand

Question 12

Can full expansion mutations be inherited from unaffected parents

Answer 12

Yes- by unaffected parents with an intermediate or permutation allele

Question 13

Does it make a difference if there is transmission of repeat instability from mother vs father

Answer 13

Yes

Question 14

What is repeat instability

Answer 14

Repeats can expand in subsequent generations

Question 15

What is ‘anticipation’

Answer 15

An intermediate repeat or a small expansion can increase in size in subsequent generations

Results in a more severe phenotype in child than in the parent

Question 16

Who is transmission of MDtype1 more likely to be from

Answer 16

Mother

Question 17

Who is more likely to have children of larger expansions of CTG triplet

Answer 17

Female carriers of unstable repeat sizes

Question 18

How does congenital DM1 occur

Answer 18

Almost exclusively via maternal transmission

Question 19

Where does expansion of CTGs take place for DM1

Answer 19

In the oocyte

Question 20

Who is large expansions of CAG repeat in Huntington’s Disease more likely passed down from

Answer 20

Father

Question 21

Who is likely to have children with larger expansions in CAG repeats in Huntington;s

Answer 21

Male carriers of unstable repeat sizes

Question 22

Who is more likely to have Greater risk of Juvenile Onset HD

Answer 22

Those with fathers who are carriers of unstable repeat sizes

Question 23

Where does expansion of CAGs in Huntington’s take place

Answer 23

Spermatogenesis

Question 24

When may a single allele be detected when using PCR to interpret HD1+HD3 results

Answer 24

• patients may be homozygous for this repeat size (i.e. inherited an allele with 17 repeats from each parent)
• Patient may have an expansion that is not detectable by this method

Question 25

When would you use HD2 and HD5

Answer 25

When you have a single gene detected when using PCR to interpret HD1 and HD3 results

Question 26

What does using HD2 and HD5 tell you and not tell you.

How would you use them

Answer 26

How to use them:
-They amplify DnA across the CAG AND the CCG repeat (cCG is one adjacent)

-HD2 primer is fluorescently labelled. PCR fragment sizes can be detected by capillary electrophoresis

What they can and can’t tell you:
Can’t be used to size the repeat but useful to confirm alleles have been detected

Question 27

If there is only one allele detected when using HD1+3 and HD2+5 tests, what does that tell you

Answer 27

Patient may be homozygous for this repeat size (i.e. inherited an allele with 17 repeats from each parent)

Patient may have an expansion that is not detectable by this method

Question 28

When is triplet prime PCR used

When is It particularly important

Answer 28

Used in HD for individuals homozygous for normal alleles on both HD1+HD3 and HD2+HD5 PCRs

Particularly important for possible cases of Juvenile HD

Question 29

What are the primers used in Triplet Primed PCR

Answer 29

HD_TPF

HD_TPR

HD Tail

Question 30

What does the HD_TPF primer do

Answer 30

Binds to specific sequence upstream of the CAG repeat

Question 31

What does the HD_TPR primer do

Answer 31

Binds to the repeat sequence (CAG)

Has a ‘tail’ with unique sequence

Question 32

What does the HD Tail primer do and its role

Answer 32

Binds to the HD_TPR tail

-Amplifies the different sized products generated

Question 33

Why can TP-PCR produce fragments of different sizes

Answer 33

Because the HD_TPR primer can bind anywhere on the repeats

Question 34

Why is HD_TPR at a limiting concentration

Answer 34

Prevents gradual shortening of product size

Question 35

What are products of TP-PCR separated by

Answer 35

Capillary electrophoresis

Question 36

how can you tell there’s an expansion present in a TP-PCR graph

Answer 36

Normal results show a clear end to the triplet peaks

Positive (expansion) results show peaks continue until they tail off