the deoxy-HbS oxy-HbS ratio should reduce the likeli-
hood of sickling and the severity of the disease. These
approaches have been tried but so far have failed to ame-
liorate the disease.
Among Bedouin Arabs and some populations of central
and southern India, high HbF levels reduce the severity of
sickle cell disease by inhibiting the formation HbS poly-
mers. This observation has led to therapeutic approaches
to induce higher levels of HbF in patients with sickle
cell disease. The therapeutic agents are hydroxyurea and
short-chain fatty acid derivatives. Hydroxyurea is an an-
tineoplastic agent that inhibits ribonucleotide reductase
(Chapter 27). It is thought that in bone marrow, hydrox-
yurea selectively kills many precursor cells but spares ery-
throblasts that produce HbF. Hydroxyurea therapy also
results in decreased circulating granulocytes, monocytes,
and platelets. These changes, along with increased HbF,
reduce vaso-occlusion due to a decreased propensity for
sickling and adherence of red blood cells to endothelium.
The long-term toxicity of hydroxyurea due to its myelo-
suppressive and teratogenic effects is not known. Induc-
tion of HbF by short-chain fatty acids such as butyrate was
discovered in infants of diabetic mothers who had a delay
in switching from HbF to HbA in association with ele-
vated serum levels of amino-n-butyrate. This led to stud-
ies of HbF induction with several short-chain acids, in-
cluding butyrate, all of which induce production of HbF.
The mechanism by which butyrate and other short-chain
fatty acids affect gene expression involves transcriptional
activation of a y-globin gene at the promoter site. One
of the mechanisms of regulation of transcription, which
occurs at the promoter site, is due to changes in histone
acetylation and deacetylation (Chapter 26). Acetylation
of specific histones by acetylases allows increased bind-
ing of transcription factors to target DNA that stimulates
transcription. Deacetylation by histone deacetylases of hi-
stones prevents transcription. Butyrate is an inhibitor of
deacetylase and it is thought that this action leads to in-
duction of y-globin gene expression. Since hydroxyurea
acts by a different mechanism in the elevation of HbF,
butyrate’s action can be additive or synergistic. Sustained
induction of HbF by pulse butyrate therapy in patients with
sickle cell anemia has resulted in up to a
% increase in
HbF along with marked clinical improvement.
Hemoglobin E results from the substitution of glutamic
acid with lysine at the 26th residue of the
chain; it has a
high prevalence in Southeast Asia. Neither heterozygous
nor homozygous states are associated with significant clin-
ical abnormalities. The homozygous state exhibits a mild
anemia, and both states show red blood cell indices resem-
bling those of heterozygous thalassemic states, namely,
hypochromic microcytosis. The latter has been attributed
to activation of a cryptic splice site in exon
of /Tglobin
mRNA caused by
mutation. This leads to abnormal
splicing of /3-globin mRNA, producing a less stable and
ineffective mRNA. Coinheritance of the HbE gene with
different forms of thalassemia genes is also known.
and 6-Globin Mutants
and 5-chain mutants are difficult to study because
of the small fraction of HbF and HbA
present in adult
erythrocytes. Overt clinical symptoms associated with
and 5-chain variants are rare. By routine screening,
14 5-globin variants have been discovered but are of no
clinical consequence. Thirty-five mutant
sequences (in-
volving the Gy or the Ay chains) have been identified.
They are all benign except for HbF Poole [Gy130(H8)
Tyr —»■
Gly], an unstable hemoglobin that causes hemolytic
disease in the newborn.
Screening and Prenatal Diagnosis
Screening programs are aimed at detection of the most
common and most harmful hemoglobin genes and the
identification of heterozygotes. Screening can provide a
basis for genetic counseling and decrease the number
of homozygous individuals conceived. Electrophoresis is
used to differentiate among sickle cell trait, sickle cell ane-
mia, and HbSC disease and to detect thalassemia, hered-
itary persistence of HbF, and HbC trait and HbC disease
(see Appendix VII). Prenatal diagnosis of genetic disease
often complements programs for detection of heterozy-
gotes. If both parents are known to be carriers, a homozy-
gous fetus can be detected early in pregnancy. The first
prenatal tests measured the ability of fetal reticulocytes
to direct the synthesis
in vitro
of normal or abnormal
/1 chains. Fetal blood was obtained either by percutaneous
aspiration from the placenta or transvaginally by direct
aspiration from one of the fetal vessels on the surface of
the placenta. The cells were incubated with radioactive
amino acids, and the labeled globin chains were separated
by chromatography and quantitated. This method detects
normal and sickle cell anemia genotypes, absence of
/f-globin synthesis (homozygous
or /T-thalassemia),
and some types of /^-thalassemia. DNA has been iso-
lated directly from cultured fetal cells (obtained by am-
niocentesis and from trophoblast biopsy) and subjected
to restriction endonuclease digestion. (Culturing of fetal
cells is necessary, since their number in the amniotic fluid
sample is small.) The restriction fragments are separated
according to size by electrophoresis and reacted with a
labeled DNA probe that is complementary to the gene be-
ing sought. The hybridization pattern is compared with
previous page 702 Bhagavan Medical Biochemistry 2001 read online next page 704 Bhagavan Medical Biochemistry 2001 read online Home Toggle text on/off