Page 119 - Essential Haematology

Chapter 7 Genetic disorders of haemoglobin / 105

form rhomboidal crystals and in the homozygous Polymerase chain reaction (PCR) is the most
state there is a mild haemolytic anaemia with commonly used technique (Fig. 7.20 ) and may be
marked target cell formation, cells with rhomboidal performed by using primer pairs that only amplify

shape and microspherocytes (Fig. 7.19 b). Th e individual alleles ( ‘ allele - specific priming ’ ) (Fig.

spleen is enlarged. The carriers show a few target 7.21 ) or by using consensus primers that amplify all
cells only. the alleles followed by restriction digestion to detect

a particular allele. This is best illustrated by Hb S
in which the enzyme DdeI detects the A - T change
Haemoglobin D d isease
(Fig. 7.22 ).

This is a group of variants all with the same electro- Gap - PCR analysis is useful for detecting gene
phoretic mobility. Heterozygotes show no haema- deletions in α - thalassaemia (Fig. 7.23 ), for δ β -
tological abnormality while homozygotes have a thalassaemia and Hb Lepore. Small deletions and
mild haemolytic anaemia. point mutations are diagnosed by cycle - sequencing
of PCR product using fluorescent labels and analy-

sis of the fragments on a capillary - based automatic
Haemoglobin E d isease
sequencer. This approach is useful for rare and

This is the most common haemoglobin variant unknown mutations, for confirming prenatal diag-

in South - East Asia. In the homozygous state, nosis of β - thalassaemia and sickle cell disorders by
there is a mild microcytic hypochromic anaemia. other PCR methods, and for the rare non - deletion
0
+
Haemoglobin E/ β - thalassaemia, however, resem- α - thalassaemia mutations that result in severe Hb
0
bles homozygous β - thalassaemia both clinically H hydrops fetalis syndrome.
and haematologically. Pre - implantation genetic diagnosis which avoids
the need for pregnancy termination involves per-
Prenatal d iagnosis of g enetic Double-stranded
h aemoglobin d isorders DNA
1)
It is important to give genetic counselling to Heat to 94ºC to
couples at risk of having a child with a major denature to
single strands
haemoglobin defect. If a pregnant woman is found
to have a haemoglobin abnormality, her partner 2) Primer Anneal single strands
should be tested to determine whether he also to synthetic
oligonucleotide
carries a defect. When both partners show an abnor- primers and
mality and there is a risk of a serious defect in the reassociate
Primer

offspring, particularly β - thalassaemia major, it is
important to offer antenatal diagnosis. Several tech- 3) Add DNA polymerase

+ dNTPs to
niques are available, the choice depending on the synthesize new
stage of pregnancy and the potential nature of the strands on template
of existing strands
defect.
4) Repeat process 20 – 30 times
DNA d iagnosis

Figure 7.20 Polymerase chain reaction (PCR). The

The majority of samples are obtained by chorionic primers hybridize to DNA on either side of the piece of

villus biopsy although amniotic fluid cells are some- DNA to be analysed. Repeated cycles of denaturation,
times used. Techniques to sample maternal blood association with the primers, incubation with a DNA
for fetal cells or fetal DNA are being developed. Th e polymerase and deoxyribonucleotides (dNTPs) results
DNA is then analysed using one of the following in amplifi cation of the DNA over a million times within
methods. a few hours.