The Thalassemias

The thalassemias are a heterogeneous group of
diseases caused by decreased or absent formation
of a globin chain. The thalassemias occur
predominantly in the Mediterranean region,
parts of Africa, and Southeast Asia (the word
thalassemia is derived from thalassa, the Greek
word for sea). In these regions, they are a significant
cause of morbidity and mortality, but they
are frequent because heterozygotes are protected
from severe malarial infection

Thalassemia

Thalassemia, a chronic anemia
Depending on which globin chain is not formed
in sufficient amounts, !-thalassemia, "-thalassemia,
or #"-thalassemia results. This leads to
chronic anemia, which causes the various manifestations
of thalassemia. Oxygen deficiency in
the peripheral tissues leads to increased extramedullary
(outside the bone marrow) blood
formation. A tendency toward infection, undernourishment,
and other signs characterize the
severe clinical picture.

!-Thalassemia and "-thalassemia

The thalassemias have a wide spectrum of
different genotypes and phenotypes (disease
manifestations and course). In the "-thalassemias
(1), complete absence ("0) is distinguished
from decreased formation ("+) of the "
chain. With the !-thalassemias (2), one, two,
three, or all four loci for !-globin may be affected.
Altogether, there are 12 principal genotypes.
In individuals with two mutations at the
!-loci (!-thalassemia), the two can lie either on
the same chromosome (thal-1) or on different
chromosomes (thal-2). Thal-1 occurs mainly in
Southeast Asia; thal-2, mainly in Africa. Each !
gene is located within a 4 kb region of homology,
interrupted by small, nonhomologous
regions. The most frequent mechanism for the
origin of a chromosome with only one !-globin
gene is nonhomologous crossing-over between
two !-globin gene loci after mispairing of the
homologous chromosomes during meiosis.

!-Thalassemia due to different mutations

Many mutations in the "-globin gene region can
lead to "-thalassemia. The mutations may also
occur in noncoding sequences (5! to exon 1 and
within introns).

Haplotypes resulting from polymorphic restriction sites in the !-globin gene cluster

From the presence or absence of recognition
sites of a number of restriction enzymes (restriction
fragment length polymorphism, RFLP),
different haplotypes can be distinguished in the
"-globin-like gene region ("-globin gene
cluster). Each haplotype is characterized by the
presence or absence of several polymorphic restriction
sites. By establishing the haplotypes of
the affected and unaffected individuals within a
family, the mutation-carrying haplotype can be
identified (indirect genotype analysis). Different
mutations have occurred on the background
of different haplotypes. Frequently, a
particular mutation is linked to a distinct haplotype
(linkage disequilibrium). This reflects the
time elapsed since the mutation first occurred
in the population, where it has been maintained
by selection.

Thalassemia

Thalassemia may be associated with mental
retardation. Two different syndromes can be
distinguished: One occurs in patients with a
large (1–2 Mb) deletion on the tip of chromosome
16 including the !-globin gene cluster
(ATR-16 syndrome). The other is an X-linked
disorder with a remarkably uniform phenotype
and a mild form of HbH disease without !-globin
deletion. A trans-acting regulatory factor
appears to be encoded on the X chromosome.

Hereditary Persistence of Fetal Hemoglobin (HPFH)

Hereditary persistence of fetal hemoglobin
(HPFH) refers to a genetically heterogeneous
group of diseases in which the temporal expression
of the !-globin genes during development
has been altered. Individuals with HPFH produce
increased amounts of fetal hemoglobin
(HbF). Under some conditions, HbF may be the
only !-globin-like gene product formed. Clinically,
HPFH is relatively benign, although HbF is
not optimally adapted to postnatal conditions.
Analysis of HPFH has yielded insight into the
control of globin gene transcription and the effect
of mutations in noncoding sequences.

Large deletions in the !-globin gene cluster

A numer of very large deletions in the !-globin
gene cluster region are known, especially in the
3! direction. The deletions show different distributions
in different ethnic populations, reflecting
that they originated at different points
in time. "!-Thalassemia and failure of !-globin
production have been the result in some cases.

Mutations in noncoding sequences of the promoter region

Mutations in the noncoding sequences of the
promoter region at the 5! end of the !-globin
cluster (on the 5! side of the #-globin genes) can
also lead to hereditary persistence of fetal
hemoglobin. Although the highly conserved
sequences CACCC, CCAAT, or ATAAA are not affected,
the number of observed mutations substantiates
the significance of the remaining
noncoding sequences (long-range transcription
control). They are probably required for the
changes in transcription control of the different
gene loci that occur during embryonic and fetal
development.

According to estimates of the WHO

According to estimates of the WHO (BullWorld
Health Org. 1983) about 275million persons are
heterozygotes for hemoglobin diseases worldwide.
Substantial numbers are due to the !-
thalassemias in Asia (over 60 million), $0-
thalassemia in Asia (30 million), HbE/!-thalassemia
in Asia (84 million), and sickle cell heterozygosity
in Africa (50million), India, the Caribbean,
and the USA (about 50 million). At least
200000 severely affected homozygotes are
born annually, about 50%, due to sickle cell anemia
and 50% to thalassemia

DNA Analysis in Hemoglobin Disorders

Numerous procedures that do not require direct
determination of the altered nucleotide base
sequence can be used to demonstrate a mutation.
An available probe of the gene or gene
region being investigated and knowledge of
the normal Southern blot pattern after restriction
analysis of the gene are prerequisites

Direct demonstration of a deletion

A partial deletion may cause an altered pattern
of a Southern blot. Two genes (!2 and !1) of !-
globin are presented (1). They are both located
on a 14.5 kb restriction fragment. If partial deletion
results in loss of a segment of, e.g., about
4.5 kb that is part of both the !2 and the !1
gene, a fragment of 10.0 instead of 14.5 kb will
result in this area (2). Three genotypes are
possible (3): two normal genes, !2 and !1, represented
by a 14.5 kb fragment; a normal DNA
segment (14.5 kb) and one with a deletion (10.0
kb); or a deletion in both gene segments (only
one fragment, of 10.0 kb). This can be demonstrated
directly in the Southern blot pattern
with a probe for the !1 gene (4).

Indirect evidence for a mutation by RFLP analysis

Indirectly, a mutation can be demonstrated if
there is an individual difference (polymorphism)
in the base sequences of the mutant and
the normal gene segments (restriction fragment
length polymorphism, RFLP, see p. 64). For
instance, if two of the same DNA segments
differ in a polymorphism for the recognition
sequence of a restriction enzyme, then DNA
fragments of different sizes (here, either 7 kb
and 6 kb, or 13 kb) result after cleavage with the
enzyme (1). If the mutation has occurredwithin
the 13 kb fragment, then this fragment indicates
presence of the mutation. In the given
DNA segment, there are three possibilities
(genotypes): two fragments of 7 kb without
mutation; one fragment of 7 kb (normal) and
one fragment of 13 kb (which carries the mutation);
and two mutation-carrying fragments of
13 kb (3). The Southern blot (4) shows whether
the person being examined is homozygous normal
(has no 13 kb fragment), is heterozygous (a
7 kb and a 13 kb fragment), or is homozygous
for the mutation (two 13 kb fragments).
The prerequisite for this indirect analysis is previous
knowledge of which of the DNA fragments
contains the mutation. The observed
difference is not the result of the mutation, as in
A. If the Southern blot pattern of affected and
unaffected individuals does not differ, then this
method will not be informative for the disorder.

Demonstration of a point mutation from an altered restriction site

A restriction site may be altered by a mutation.
For example, a sickle cell mutation in codon 6 of
the " gene of hemoglobin (see p. 340) (1) causes
loss of a restriction site for the enzyme MstII
(CCTNAGG instead of CCTNTGG) because the A
(adenine) has been replaced by a T (thymine)
(2). The normal allele ("A) in this area produces
a 1.15 kb fragment after MstII digestion,
whereas the mutation eliminates the restriction
site in the middle so that a 1.35 kb fragment
results. The 1.35 kb fragment in the Southern
blot indicates (3) the presence of the sickle cell
mutation ("S). Thus, homozygous normal individuals
(AA), heterozygotes (AS), and homozygotes
for the sickle cell mutation (SS) can
be clearly distinguished; each of the three genotypes
can be precisely diagnosed.

Peroxisomal Diseases

Peroxisomes are small round organelles about
0.5–1.0 μm diameter (somewhat smaller than
mitochondria). They are found mainly in the cytoplasm
of kidney and liver cells. They are the
site of some important metabolic functions. The
name is derived fromhydrogen peroxide, which
is formed as an intermediary product of oxidative
metabolism in the peroxisomes. A number
of defects in peroxisome formation or peroxisome
enzymes lead to severe diseases in
humans (peroxisomal diseases).

Biochemical reactions in peroxisomes

The electron micrograph (1) shows peroxisomes
in a section of rat liver. The dark striated
structures within the organelles consist of
urates (peroxisomes contain an enzyme that
oxidizes uric acid). Peroxisomes have both catabolic
(substances are degraded) and anabolic
(substances are synthesized) functions (2). Two
biochemical reactions are especially important:
a peroxisomal respiratory chain and the !-oxidation
of very long-chain fatty acids. In the peroxisomal
respiratory chain (3), certain oxidases
and catalases act together. Specific substrates of
the oxidases are organic metabolites of intermediary
metabolism. Very long-chain fatty
acids are broken down by !-oxidation (4) in a
cycle with four enzymatic reactions. Energy
production in peroxisomes is relatively inefficient
compared with that of mitochondria.
While free energy in mitochondria is mainly
preserved in the form of ATP (adenosine triphosphate),
in peroxisomes it is mostly converted
into heat. Peroxisomes are probably a
very early adaption of living organisms to oxygen.

Peroxisomal diseases

Several peroxisomal diseases are known in
man; the six most important are listed. All are
autosomal recessive hereditary disorders.
Patients with neonatal adrenoleukodystrophy
do not form sufficient amounts of plasmalogens
and cannot adquately degrade phytanic acid
and pipecolic acid. When cultured fibroblasts
frompatientswith genetically different types of
peroxisomal diseases are fused, the hybrid cells
form normal peroxisomes (cells with different
defects can correct each other).

Cerebro-hepato-renal syndrome type Zellweger

Patients with this autosomal recessive hereditary
disease have a characteristic facial appearance
(1–4), extreme muscle weakness (5), and
a number of accompanying manifestations such
as calcified stippling of the joints on radiographs
(6), renal cysts (7, 8), and clouding of the
lens and cornea. The severe form of the disease
(type Zellweger) usually leads to death before
the age of one year.