Molecular Diagnosis of Huntington's Disease

Question 1

From whom will diagnostic HD test requests be accepted?

Answer 1

• Diagnostic testing in adults will be accepted from Neurologists, Psychiatrists, specialists in care of the elderly.

Question 2

Why must diagnostic HD for minors go through clinical genetics?

Answer 2

Diagnostic testing of minors (<16yrs) must be referred to the clinical genetics dept to ensure that appropriate counselling has been received by both the patient and the parents. A positive result for the child is essentially a predictive result for one of the parents if they are unaffected at the time.

Question 3

Why won’t HD referrals be accepted from GPs?

Answer 3

Referrals from GPs are not tested as patients may not have been appropriately counselled about the implications of HD diagnosis.

Question 4

When may confirmation of diagnosis of HD be requested?

Answer 4

Confirmation of the diagnosis of HD may be requested where a relative is known to be affected with HD. A copy of the report and a control sample should be obtained for that family member if possible.

Question 5

What types of HD referrals may the lab receive?

Answer 5

1) . Diagnostic testing in adults from neurologists, psychiatrists, specialist in care of the elderly.
2) . Confirmation of a diagnosis of HD.
3) . Presymptomatic testing via clinical genetics.
4) . Prenatal diagnosis via clinical genetics which may be a direct test or performed via exclusion testing.

Question 6

All prenatal samples must also be checked for what?

Answer 6

MCC

Question 7

What is the direct method of prenatal testing?

Answer 7

Determining the size of the CAG repeat if the relevant parent already knows their CAG status.

Question 8

How is CAG repeat sizing typically carried out?

Answer 8

The Cy5-CAG PCR is typically used for CAG repeat sizing.

CAG repeat sizing is typically performed using 2 sets of primers flanking the CAG repeat region, HD1A + HD3 and HD1alt + HD2. This eliminates problems caused by primer binding site polymorphisms.

One primer in each PCR is fluorescently labelled and the two PCRs are carried out together due to the 10 day reporting time for diagnostic and predictive HD referrals.

PCR products are separated by capillary electrophoresis..

Internal controls are used with known allele sizes. This allows patient samples to be compared in order to accurately size the CAG repeats.

PCR across the CAG repeat region using primers HD1A and HD3 is used to establish the CAG repeat length by comparison with size controls.

The HD1alt and HD2 PCR is used to resolve patients with 2 normal alleles of the same size.

Question 9

What 2 sets of primers are usually used for CAG repeat sizing?

Answer 9

1) . HD1A+HD3

2) . HD1alt+HD2

Question 10

Describe the HD1A + HD3 PCR.

Answer 10

The HD1A + HD3 PCR is used for accurately sizing CAG repeat expansions.

The HD1A primer has been adapted from the HD1 primer described by Warner et al. in 1993 to avoid a rare polymorphism at the 3’ end which causes allele dropout.

A rare polymorphism in the HD3 primer can also potentially disrupt primer binding and result in a false negative result so care must be taken when interpreting results.

The HD1alt + HD2 PCR would show an expansion even if the HD1A+HD3 PCR showed an apparently negative result.

The observation of a single allele from this assay may therefore represent:
1). A true homozygote
2). A normal allele and an unamplifiable large expansion
3). A normal allele and a small expansion that has not been amplified due to a polymorphism under the HD3 primer.
This can be resolved using the second set of primers, HD1alt and HD2.

In referrals from children and young adults a positive control of >80 CAG repeats should always be used.

Question 11

What PCR is used to accurately size CAG repeat expansions?

Answer 11

The HD1A + HD3 PCR is used for accurately sizing CAG repeat expansions.

Question 12

In Cy5-CAG PCR how is the allele size calculated?

Answer 12

Allele size is calculated by comparison to positive controls of known allele size. The highest peak is selected as the allele size.

Question 13

What kind of Cy5-CAG PCR profile results might we expect to see for larger alleles?

Answer 13

Larger alleles show a hegehog profile.

Allele size is determined from the highest peak plus or minus the number of peaks to the right of the highest one.

Question 14

Describe the HD1alt + HD2 PCR.

Answer 14

The CAG repeats in the HD gene are immediately 5’ of a CCG repeat which is also polymorphic in length.

Primers HD1alt and HD2 encompass both the CAG and the CCG repeat region.

Incorporation of the CCG repeat region enables two normal alleles to be resolved in cases where both alleles have identical numbers of CAG repeats, but different numbers of CCG repeats (in the absence of primer site polymorphisms).

Thesis primers are not used to determine CAG repeat length.

Question 15

What can’t the Cy5-CAG PCR detect?

Answer 15

The Cy5-CAG PCR can detect all normal alleles and most disease-associated expanded alleles in adults (have detected alleles of approximately 85 and approximately 115 repeats).

Additional testing procedures are required if patients are homozygous for a normal allele in both PCRs, especially important in cases of suspected juvenile HD.

Techniques to detect larger alleles include triplet primed PCR (TP-PCR) and Southern blotting.

Question 16

What techniques can be used to detect larger alleles that Cy5-CAG PCR won’t necessarily pick up?

Answer 16

1) . Triplet primed PCR (TP-PCR)

2) . Southern blotting

Question 17

Briefly describe the triplet-primed PCR (TP-PCR) method.

Answer 17

TP-PCR is used to exclude the presence of a large expansion that won’t amplify well with a standard PCR.

This technique is typically used when only 1 normal allele can be detected by both standard PCRs, or when alleles of only 1 triplet apart are detected by HD1A + HD3 and a single allele is detected by the HD1alt + HD2 PCR.

A patient with 2 alleles of the same size should show no periodicity or no large peaks and noise in the repeats expansion region. However, a patient with alleles of different sizes will show some periodicity, with humps representing each allele.

TP-PCR is always performed with a large size control. In diagnostic cases where the onset of symptoms has occurred at a relatively late age it is unlikely that the patient would have a large expansion.

Data reported by Brinkman et al. suggests that all individuals with an expansion of 50 CAGs will have the onset of HD by the age of 35.

The TP-PCR is a 3 primer system. The forward P1 primer is fluorescently labelled for capillary electrophoresis (Cy5). This primer binds to the sequence upstream of the repeat. The reverse primer (P4) binds to CAG repeats at multiple locations. However, the 5’ end of the primer is non-specific. P4 is at a limiting concentration so that it is exhausted after the first few PCR cycles. This prevents priming from the PCR products that would lead to gradual shortening of product size. The tail primer (P3) is specific to the 5’ end of the reverse primer. The tail primer allows further amplification of the products of the forward and reverse primers. The final pool of products represents the whole repeat length, giving a distinct trace after capillary electrophoresis.

Question 18

When is TP-PCR likely to be used?

Answer 18

Used in HD for individuals homozygous for normal alleles on both PCRs.

This technique is typically used when only 1 normal allele can be detected by both standard PCRs, or when alleles of only 1 triplet apart are detected by HD1A + HD3 and a single allele is detected by the HD1alt + HD2 PCR.

Particularly useful in possible cases of juvenile HD.

Question 19

How should a patient with 2 alleles of the same size appear on TP-PCR?

Answer 19

A patient with 2 alleles of the same size should show no periodicity or no large peaks and noise in the repeats expansion region. However, a patient with alleles of different sizes will show some periodicity, with humps representing each allele.

Question 20

Describe the functions of the primers used in TP-PCR.

Answer 20

The TP-PCR is a 3 primer system.

The forward P1 primer is fluorescently labelled for capillary electrophoresis (Cy5). This primer binds to the sequence upstream of the repeat.

The reverse primer (P4) binds to CAG repeats at multiple locations. However, the 5’ end of the primer is non-specific. P4 is at a limiting concentration so that it is exhausted after the first few PCR cycles. This prevents priming from the PCR products that would lead to gradual shortening of product size.

The tail primer (P3) is specific to the 5’ end of the reverse primer. The tail primer allows further amplification of the products of the forward and reverse primers.

The final pool of products represents the whole repeat length, giving a distinct trace after capillary electrophoresis.

Question 21

What is one of the main advantages of TP-PCR over Southern blotting?

Answer 21

It is a much faster method.

Question 22

What is one of the limitations of TP-PCR?

Answer 22

It does not size the expansion.

Question 23

What is an alternative method to TP-PCR to detect large expansions?

Answer 23

Use a Southern blotting approach.

Question 24

Describe a Southern blotting approach to detect large repeat expansions.

Answer 24

DNA samples are digested with the restriction enzyme Pst1. Agarose gel electrophoresis separates out the digested products which are then transferred to a nitrocellulose membrane using the standard Southern blotting techniques. Typically, radioactive hybridisation of the 4GP1.7 probe is utilised to detect the differently sized alleles. Normal and positive controls are included on the blot and so any expansion can be compared and sized.

Question 25

What further analysis can be used to support the results of the Cy5-CAG PCR/TP-PCR results?

Answer 25

HD Linkage analysis can be used to support the results of the Cy5-CAG PCR/TP-PCR results - particularly for prenatal diagnosis.

Linkage analysis is the only method that can be used for exclusion testing.

This technique is dependant on the family structure and availability of DNA from relevant family members.

Polymorphic markers in the HTT region on chromosome 4p16.3 can be used. Ideally markers for both proximal and distal to the HTT gene need to be informative to have a secure result.

A multiplex of 9 microsatellite markers that flan the HTT gene can be used to determine inheritance of the low or high risk allele. It is advisable to determine which markers are informative for a particular family before a prenatal exclusion test is undertaken. A pedigree with the result of the haplotype analysis should be included in the report.

Question 26

What analysis is relied upon for HD exclusion testing?

Answer 26

Linkage analysis.