laboratory demonstration of the deficiency. If the
deficiency is due to the production of a hypofunc-
tional or nonfunctional protein, the mass concentra-
tion of the protein may be normal, so the deficiency
must be demonstrated using a functional assay. If
the deficiency results from decreased protein synthe-
sis, decreased protein secretion, or an increased
plasma clearance rate of the protein, both the
functional activity of the protein and its mass
concentration will be decreased. In these conditions,
the deficiency can be demonstrated using either a
functional assay or a mass assay. Functional protein
assays measure the specialized function of the
protein. Mass assays for the plasma proteins are for
the most part based on immunologic methods.
Additionally, semiquantitative measurement of the
mass concentration of two plasma proteins,
α
1
-anti-
trypsin and IgG, is possible using protein electro-
phoresis. The
α
1
and
γ
fractions of plasma consist
almost entirely of
α
1
-antitrypsin and IgG, respec-
tively, so the intensity of each band when visualized
with a protein stain is proportional to the mass con-
centration of the respective protein.
The foregoing approach cannot be used in the
evaluation of all plasma protein deficiencies, how-
ever. For instance, in the case of
α
1
-antitrypsin
deficiency, measurement of plasma
α
1
-antitrypsin
concentration, whether by functional or mass assay,
can be diagnostically unreliable because
α
1
-anti-
trypsin is an acute phase reactant. Its plasma con-
centration can increase several-fold over baseline
values in response to infections and inflammatory
conditions. Hence, even patients with severe
α
1
-antitrypsin deficiency can have low normal
protein concentrations during an acute phase
reaction.
A more reliable approach for the diagnosis of
α
1
-antitrypsin deficiency is to characterize the pheno-
type of the patient’s
α
1
-antitrypsin using a pheno-
typic marker that distinguishes the proteins produced
by the major alleles for the protein. This approach
is referred to as protein phenotyping. The marker
employed in the protein phenotyping of
α
1
-antitrypsin is the isoelectric point of the protein as
revealed by isoelectric focusing on acrylamide gels
(Jeppsson and Franzén 1982).
The most common alleles for
α
1
-antitrypsin are
the M alleles. The M allele products have normal
elastase inhibitory function and are present in normal
concentration in the plasma. The common allelic
variants that cause
α
1
-antitrypsin deficiency are the S
allele and the Z allele (Norman
et al.
1997). The S
allele product (264 Glu
→
Val) is functional and is
synthesized in normal quantities but undergoes accel-
erated intracellular degradation so that subnormal
amounts are secreted. Homozygous individuals have
plasma
α
1
-antitrypsin concentrations that are about
half normal. The Z allele product (342 Glu
→
Lys)
is somewhat hypofunctional. It is synthesized in
normal quantities but tends to polymerize in the
endoplasmic reticulum so that only small amounts
are secreted. Individuals homozygous for the Z
allele have plasma
α
1
-antitrypsin concentrations that
are 10 to 15 percent of normal.
The amino acid substitutions of the S and Z
alleles result in
α
1
-antitrypsins with isoelectric points
that are different from the normal M allele product
and from one another. Examination of the band pat-
tern of a sample following isoelectric focusing
reveals the
α
1
-antitrypsin phenotypes present in the
plasma and thereby allows the genotype of the
patient to be inferred (Figure 10.5).
When there are numerous allelic variants for a
protein, it is generally not possible to find a practical
phenotypic marker that can distinguish all of the
variant forms of the protein. It is usually considered
adequate that the phenotype succeed in identifying
the most common variants.
Genetic disorders of blood cells
The proteins involved in the genetic disorders of
blood cells encompass enzymes of cellular metab-
olism, structural proteins of the cytoskeleton and the
membrane, and specialized proteins of cell function.
Disorders that serve as examples from the red cell
lineage are, respectively, glucose-6-phosphate dehy-
drogenase (G6PD) deficiency, hereditary spherocy-
tosis, and sickle cell anemia. Many of the genetic
disorders of blood cells can be diagnosed with
molecular diagnostic studies but, at the present,
these studies are used mostly for prenatal diagnosis
(discussed later in this chapter). Postnatal diagnosis,
which is usually pursued in childhood, depends, for
the most part, upon laboratory approaches appropri-
ate to the involved protein. For instance, disorders
of the enzymes of cellular metabolism are evaluated
using i
n vitro
assays of blood cell enzyme activity.
Genetic disorders of structural proteins may be
diagnosed by the characteristic morphologic abnor-
malities that result from the deficient or dysfunc-
tional protein product. Alternatively, i
n vitro
functional studies may reveal function deficiencies
Genetic Disease
10-9
