section 28.4
Derivatives of Hemoglobin
Flanking raglon
globln gene
5 8.7
’ ’*
l 02 I
---------------------------------- 'vsii
Ms) It fragment
FIGURE 28-16
Diagram of the 5' region of the human /S-globin gene and its flanking
region, showing the cleavage sites of the restriction endonuclease Mst II
(vertical arrows). The fragments obtained by digestion of normal and
sickle /S-globin genes are shown in the lower part of the diagram. Shaded
regions of the gene are the exons, and IVS indicates the introns
(intervening sequences). [Reproduced with permission from J. C. Chang
and Y. W. Kan, A sensitive prenatal test for sickle cell anemia.
N . E ngl. J.
M ed.
307, 30 (1982).]
one from a normal individual (Chapter 23). Differences
between the normal and some mutant globin genes at
recognition sites for restriction endonucleases will cause
differences in the cuts made and, hence, in the sizes of the
fragments detected.
In the ideal situation, a restriction endonuclease recog-
nizes a site in the DNA that includes the mutated base.
Restriction enzymes Mnl I, Dde I, and Mst II recognize
part of the coding sequence for amino acids 5, 6, and 7 of
the normal
-globin gene. The mutation in the
gene in HbS abolishes these recognition sites, converting
two small pieces of DNA found in the digest of normal
/3-gIobin DNA into one larger fragment (Figure 28-16).
Mst II is the most useful enzyme because it generates
larger, more easily detected fragments. Loss of recognition
sites due to deletions is seen in most «-thalassemias and
occasionally in
-thalassemias, since most /3-thalassemias
are not caused by deletion. In gene deletion, multiple
sites are lost. However, point mutations are more com-
mon and therefore of greater importance than deletions.
The endonuclease that is most useful in the detection
of each disorder must be carefully selected. Because
/3-thalassemias are caused by many different mutations
with similar phenotypic expressions, it is not possible to
find one or even a few restriction endonucleases that can
detect all /1-thalassemia genes. What has been accom-
plished so far is the detection of affected individuals among
members of the same kindred or racial group in which a
single type of
-thalassemia occurs.
Alternatively, sites adjacent to an abnormal gene may
differ from those on normal chromosomes. Such linked
polymorphisms have been found for the sickle cell gene
and for several
-thalassemias. Because different individ-
uals having a particular hemoglobin abnormality will not
always have the same linked polymorphism, this method
suffers from lack of sensitivity. Under proper conditions,
short, synthetic oligonucleotide probes, complementary
to the normal DNA sequence in the region that contains
the mutation, hybridize with completely homologous se-
quences but not with sequences that differ by as little
as one nucleotide. This method is potentially more pow-
erful than restriction endonuclease mapping because an
oligonucleotide probe can be synthesized for any muta-
tion once its sequence is known. Furthermore, multiple
mutations can be detected simultaneously by hybridizing
the DNA with a mixture of different probes, making this
technique useful for screening. This technique has been
used successfully to detect the sickle cell gene and several
-thalassemias that could not be detected by restriction
endonuclease mapping.
All these methods depend on a satisfactory sample being
collected from the fetus. Studies of globin chain synthe-
sis require an adequate volume of fetal blood. Collection
methods result in 4-6% fetal death under optimal condi-
tions. Furthermore, /3-globin synthesis cannot be detected
until about the middle of the second trimester. Use of am-
niotic fluid cells obtained by amniocentesis is less dan-
gerous. However, an adequate volume of fluid cannot be
obtained until the middle of the second trimester of preg-
nancy (14-20 weeks); if culturing of the cells is necessary,
there is an additional delay of 1-2 weeks. Chorionic villus
biopsy can be obtained at about 8-12 weeks of gestation.
This allows more time for deliberation regarding termina-
tion or continuation of the pregnancy, thereby increasing
the margin of safety for the mother, should termination be
desired. This technique is no more dangerous than either
of the other methods. Some other techniques for labora-
tory evaluation of hemoglobin disorders are presented in
Appendix VII.
28.4 Derivatives of Hemoglobin
Carbon Monoxide-Hemoglobin
Carbon monoxide, an odorless gas, has an affinity for
hemoglobin that is 210 times that of oxygen. Thus, in
the equilibrium reaction
Hb02 + CO ^ HbCO + 0 2
ATeq = 2.1xl02
the equilibrium lies far to the right. Like oxygen, CO
binds to the sixth position of the heme iron. When CO and
0 2 are present together in appreciable quantities, CO is
bound preferentially and oxygen is excluded, effectively
causing an anemic hypoxia. Even if some 0 2 is bound to
the hemoglobin, it cannot be released owing to the tight
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