Using appropriate examples, discuss the relevance and application of molecular

diagnostic tests in clinical biochemistry.

Introduction
· Most genetic diseases are rare.
· Only one or two centres may test for a particular disease.
· If a single mutation accounts for the majority of disease, detection of the DNA change
may be quicker and simpler than clinical/biochemical tests
o (Haemochromatosis, AAT and apoE genotyping).

Molecular Diagnostic Tests and examples

- PCR (Polymerase Chain Reaction)
o PCR enables rapid amplification of a defined DNA sequence.
o The method involves thermo-cycling through three temperatures:
§ Denaturation (~95oC) to separate double stranded DNA template into
component single strands;
§ Annealing (~65oC) to allow synthetic DNA or oligonucleotide primer
(20bp) to pair with its complimentary single stranded DNA sequence;
§ Extension (~72oC) to allow primer extension at the 3’ end by thermally
stable DNA polymerase from Thermus aquaticus (Taq) to synthesise a
complimentary copy of the single stranded DNA template.
o PCR is a powerful diagnostic tool used to amplify specific regions of genes
containing known mutations or polymorphisms, related to specific disease.
o PCR products can be used for many downstream applications such as restriction
enzyme analysis (if mutation affects restriction site), and sequencing to
identify the mutation responsible for causing the disease.
o PCR can also be designed to directly identify the causative mutations, such as in
ARMS, Gap-PCR or TaqMan RT-PCR
o PCR is sensitive (small amount of template needed), fast and amenable to high
throughput, but it has low sensitivity (contamination) and allele drop out may
occur.
- RFLP (Restriction Fragment Length Polymorphism)
o RFLP uses PCR to amplify a sequence of interest. The PCR products are then
digested with restriction endonucleases, which cut double stranded DNA at
specific recognition sites, generating DNA fragments of different lengths.
These are resolved by agarose gel electrophoresis.
o Disease producing mutations can abolish or create restriction sites in the DNA
sequence. The pattern of digestion products can therefore determine wild type
from mutant DNA by the size of the fragments produced.
o This technique is used for detecting:
§ Sickle cell mutations. Dde1 cuts DNA specifically at C|TGAG. In sickle
cell the normal restriction site is abolished. (can also used TaqMan or
ARMS for sickle cell)
§ Primary hyperoxaluria type 2. BsmF1 site is abolished.
- ARMS (Amplification Refractory Mutation System)
o Uses PCR to selectively amplify normal and mutant alleles, based on the
specificity of primer extension. Two oligonucleotide (antisense) primers
identical in sequence, except for the 3’ nucleotides (one complementary to the
normal DNA sequence and the other to the mutant sequence). Both primers
are paired with a common sense primer. Two reactions are carried out (one for
 the normal allele and one for the mutant allele). For a primer to be extended by
the DNA polymerase the 3’ nucleotide has to be perfectly matched. The
mismatched 3’ nucleotide will not anneal to the genomic DNA template and
therefore no amplification will occur. A second pair of primers is added to
amplify DNA from an independent gene as a positive amplification control.
o ARMS is used for the detection of single base alterations. It is quick to perform
giving a qualitative result after analysing PCR product size.
o It can be multiplexed.
o ARMS is used for:
§ Alpha 1 Antitrypsin (presence of the Z and S allele)
§ Cystic fibrosis mutation analysis (~50 mutations)
§ Beta-thalassaenia
§ Hb mutation confirmation (after alpha and beta globin gene sequencing).
Large deletions in alpha thalasseamia are not picked up by sequencing,
due to lack of amplification (beta-thal is rarely caused by deletions and
cases are picked up on sequencing).
- TaqMan Real Time-PCR
o TaqMan RT-PCR uses primers to amplify a target gene sequence. A fluorescent
hybridisation probe (allele specific) then binds downstream of a primer. The
probe contains a quencher, preventing the probe fluorescing when there is a
mismatch between primer and DNA sequence. When there is a match Taq
DNA polymerase cleaves (5’à3’exonuclease activity) the fluorophore from the
probe (separated from the quencher) and allows fluorescence to occur. The
fluorescence emitted identifies the allele present (detects mutations) in the
sample.
o TaqMan assays are preferred. RT-PCR is faster than conventional PCR and
more sensitive. It shows detection of products as they form and there is no
need for gels. It uses fluorescent labels and allows quantification of product.
o TaqMan RT-PCR is used for:
§ Hereditory Haemochromatosis (HFE gene: C282Y, H63D, S65C
detection). The normal gene product is a 321aa cell surface protein
which forms a heterodimer with beta2 microglobin, which binds the
transferrin receptor. The abnormal protein does not migrate to the cell
surface and there is a lack of internalisation of transferrin.
§ HbS and HbC mutations
- Repeat sizing PCR
o Mutations can cause a size variation (insertions) in the PCR product. This can be
detected using fluorescent tags on primers to detect fragment size.
o Microsatellites (genome repetitive sequence) vary between individuals and can
be used as polymorphic markers (21-hydroxylase deficiency)
o Gilbert’s Syndrome: an insert in the TATA box repeat in the UDP-glucuronsyl
transferase 1A (7 instead of 6). This 2bp difference is detected using an
automated DNA sequencer (hard to detect 2bp difference on an agarose gel
due to the poor resolution).
- Gap-PCR
o Detects deletions in the gene sequence due to the size of the fragment detected.
o Alpha-thalassaemia: Deletion (up to 3000bp) MPLA/Multiplexing
- SSCP (Single Strand Conformational Polymorphism)
o Mobility shift of single stranded DNA in non-denaturing PAGE. This is cheap
and requires no specialist equipment. Samples denatured at 95oC (formamide),
 cooled on ice and run. Mobility depends on the primary structure and
secondary formation. Sens. ~70%.
- Denaturing HPLC (heteroduplex analysis)
o Double stranded DNA with a miss match between the strands detected by
electrophoresis or HPLC (heteroduplex elutes earlier than homoduplex). This
has an expensive outlay (dedicated HPLC system) but is quick and suitable for
high throughput.
- Sequencing (Sanger dideoxy)
o PCR in the presence of regular nucleotides and a fraction of
dideoxyribonucleotides (no 3’ hydroxyl group), which once added to the end
of a DNA strand there is no way to continue elongating. In dye-terminator
sequencing, each of the four dideoxyribonucleotide chain terminators is
labelled with a fluorescent dye with different wavelengths of fluorescence and
emission. 5% of the time a dideoxynucleotide-T will be incorporated and stop
elongation. This is the same for C, G and A. Each strand will get stopped in a
different position at random. An automated DNA sequencer uses capillary
electrophoresis to monitor the different fluorescent peaks. The fragments are
piped through a tiny glass-fibre capillary during the electrophoresis step. They
come out the far end in order of size. A UV laser built into the machine that
shoots through the liquid checks for pulses of fluorescent colours to emerge.
o Porphyria
o Thalassaemias
- Pyrosequencing
o Real time sequencing by synthesis. One dNTP added at a time, if
complementary it’s added and PPi released. PPi produces ATP used by
luciferase to produce oxyluciferin. The light produced is measured.
- Next generation sequencing
o Sequence whole genome
- Microarrays
o Analysing gene expression (small membrane containing samples of many genes
arranged in a regular pattern. Oligonucleotides attached to a silicon chip act as
specific probes and label sample with fluorescent markers) – gene loss,
comparison of two tissues
o Used for: Duchenne Muscular Dystrophy
- Southern Blot
o Gene probe with an enzyme label used to detect a gene. Detection is with X-ray
film. It can be combined with restriction enzymes.
o Used for: Duchenne Muscular Dystrophy (Large deletion)

Relevance and application

- Preimplantation diagnosis
o Delta F508 in CF
- Prenatal diagnosis
o Chorionic villus biopsy at 10-12 weeks or amniocytes 16 weeks
o The gene does not need to be expressed.
o Risk of miss-diagnosis if disease is incorrectly diagnosed in the index case.
- Carrier testing and postnatal testing
o EDTA whole blood and buccal cells
- Maternal plasma for fetal DNA.
- Population Screening
 o Fatal or chronic disability
o Treatable
o Common
- Neonatal screening
o CF (increased IRT and genetics)
o 21-hydroxylase (increased 17OHP and genetics
- Cancer Screening
o Inherited breast cancer (BRCA1 and 2)
- Can use either:
o Linkage (Uses a DNA marker to track the disease carrying chromosome within
the family. This assumes that the diagnosis in the index case is correct and the
parents are the genetic parents.)
o Mutation analysis (Detects actual cause of disease. Therefore less risk of error.
This is only possible when few mutations cause majority of disease (70%
ΔF508 in CF) or if family specific mutation known).

Ethics
· Untreatable disorder
· Burden of knowledge of carrier status
· Some countries screen for thalassaemia mutations before marriage