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Hereditary DiseasesЧCauses Cures and Problems.

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Volume 12 Number 1
January 1973
Pages 1- 90
International Edition in English
Hereditary Diseases- Causes, Cures, and Problems
By Roscoe 0. Brady‘*’
The nature of the biochemical abnormalities in over a hundred inherited diseases of humans
is now convincingly established. This impressive achievement has provided numerous practical benefits, amongst which are: 1. accurate methods for the diagnosis of patients with these
diseases ; 2. identification of symptom-free heterozygous carriers of the defective genes ;.and
3. procedures for the antenatal detection of a large number of these disorders in fetuses. It is
anticipated that equally impressive advances will be made in the future regarding the therapy
for patients with these diseases who escape prenataI detection, or in cases when an affected
fetus is identified but is carried to term in compliance with the decision of the parents. The
current concepts regarding the therapy of heritable metabolic diseases are outlined and potential innovations for future corrective procedures are indicated in this report.
1. Introduction
At the onset of the twentieth century, Sir Archibald Garrod
began his classic studies on alcaptonuria, a human disease
characterized by the gradual darkening of urine when
allowed to stand at an alkaline pH, gray to bluish black
pigmentation of the patient’s tissues, and arthritis which
begins at an unusually early age. Garrod analyzed the urine
of these individuals and clearly demonstrated that they
excreted large quantities of homogentisic acid which was
present in only trace amounts in normal human urine“’.
He discovered that this disorder occurred in various relatives of the patient. He also showed the same familial
penetrance of other human diseases which included : a)
albinism which is characterized by a failure of pigment
formation; b) pentosuria, in which there is an increased
quantity of pentose in the urine; and c) cystinuria, in which
patients have an abnormally high level of urinary cystine.
From these observations, Garrod derived the remarkable
conclusion that these individuals suffered from an inherited
deficiency of an enzyme required to catalyze a specific
metabolic step in each of these diseasesr2! He correctly
predicted the lack of homogentisic acid oxidase in alcaptonuria (Fig. 1).Some 50 years later, the correctness of Garrod‘s
hypothesis was shown by La Du et aLr3].
H o m o g c n t i s in IC
Fig. 1. Homogentisic acid is normally transformed into maleylacetoacid) by the action or
acetic acid (3,5-dihydroxy-2,4,6-octatrienedioic
homogentisic acid oxidase. The enzyme is lacking in alcaptonuria and
n o degradation takes place.
[*] Dr. R 0 Brady
Developmental and Metabolic Neurology Branch,
National Institute of Neurological Diseases and Stroke,
National Institutes of Health,
Bethesda, Maryland 20014 (USA)
Angew. Chem. biternat. Edit. / Vol. 12 (1973) / N o . 1
Inborn errors of metabolism are directly involved in more
than 100 human diseases. These disorders include ab1
normalities of amino acid metabolism such as phenylketon uria, histidinemia, and hyperprolinemia ; derangements of purine metabolism as in orotic aciduria, xanthinuria, and the Lesch-Nyhan syndrome; abnormalities of
carbohydrate metabolism such as fructosuria, galactosemia
and glycogen storage diseases ;derangements of porphyrin
metabolism ; abnormalities of protein synthesis as in sickle
cell disease, agammaglobulinemia and abetalipoproteinemia; and heritable disorders of lipid metabolism such as
Gaucher’s disease and Tay-Sachs disease.
It is not feasible to discuss or even list all of the known
human disorders of metabolism in the present contribution.
Excellent standard texts are available in which these
disorders and their clinical manifestations are catal0gued1~3~’.
It is my intention to indicate in this communication how some of the extremely perplexing problems
involved in the discovery of the nature of the abnormal
enzymology in such diseases have been resolved and to
describe some of the practical benefits which resulted from
these investigations. It will also be my task to provide a
summary of various forms of therapy which seem to be
useful at the present time and to suggest avenues of approach which may be helpful in the foreseeable future.
I shall deal with lipid storage diseases to a major extent
because a number of important, universally applicable
concepts were established during investigations of the
pathogenesis and management of these disorders.
Table 1. Salient clinical features of the sphingolipidoses
Gaucher’s disease
Mental retardation (infantile form only);
enlarged liver and spleen; hip and long
bone erosion; lipid-laden cells in bone
marrow which also stain for carbohydrate;
increased serum acid phosphatase; mild
anemia; and decreased blood platelets.
Niemann-Pick disease
Generally similar to Gaucher’s disease;
30%with cherry-red spot in macula; foam
cells in bone marrow which stain for lipid
and phosphorus; severe emaciation.
Globoid leukodystrophy
Mental retardation; almost total absence
(Krabbe’s disease)
of myelin; severe scar formation in the
brain ; multinucleated “globoid bodies”
in white matter.
Mental retardation ;psychological disturbleukodystrophy
ances; decreased nerve conduction time;
yellow-brown droplets in nerve fibers
when stained with cresyl violet (metachromasia).
Ceramide lactoside
Slowly progressing brain damage; large
liver and spleen; anemia; decreased number of white blood cells and platelets.
Fabry’s disease
Reddish-purple raised rash on abdomen
and scrotum; kidney damage; corneal
opacities; peripheral nerve pain and electrocardiographic abnormalities.
Tay-Sachs disease
Mental retardation; blindness; cherry-red
spot in the retina; enlarged skull; nerve
cells distended with “membranous cytoplasmic bodies.”
Generalized gangliosidosis Mental retardation ; cherry-red spot in
macula (50% of patients); large liver and
spleen ; foam cells in bone marrow; rarefaction of bones and skeletal deformities.
Progressive mental degeneration; weakness and spasticity of muscles; emaciation ;
thickening of the skin and enlargement of
the heart.
2. Sphingolipidoses
2.1. General Description
The are now ten distinct disorders of lipid metabolism for
which the etiology is convincingly established. The principal
clinical manifestations of these diseases are summarized in
Table 1. The hallmark of each of these disorders is the
accumulation of particular lipids which have a portion of
their molecular structure in common. This shared moiety
is called ceramide (N-acyl sphingosine).
These sphingolipids are primarily localized in membranous
elements of the cells throughout the body. One or more
molecules of carbohydrate or a phosphorylamine residue
is attached to the primary hydroxy group of the sphingosine
(Table 2).
Except for Fabry’s disease, all of the sphingolipid storage
diseases are inherited as autosomal recessive genetic
mutations. This means that both parents must be carriers
of the defect (heterozygotes)in order to produce an affected
child. Carriers are usually entirely free of the signs and
symptoms of the disorders. When both parents are carriers,
one of four children will have the disease on a statistical
basis. Such a child is called a homozygote. Two of their
children will be heterozygotes like the parents, and one
child will not be involved at all.
The genetic transmission of ,Fabry’s disease is on the X
chromosome, and the disorder is therefore carried by the
female. Half of the sons born to a female carrier of Fabry’s
disease will have the full-blown syndrome of Fabry’s
disease. Fifty percent of her daughters will be carriers, and
the other daughters will not be involved. Unlike other
sphingolipidoses,the female Fabry heterozygotes may have
some of the clinical manifestations of the disease although
they usually are much milder than those found in an afflicted male.
The underlying metabolic defect in all of the sphingolipid
storage diseases is an attenuation or complete absence of
a specific hydrolytic enzyme required for the catabolism
of a lipid which occurs in the body as a consequence of the
normal turnover of cells and tissues. A second important
fact is that the degree of enzyme deficiency is similar in all
of the organs and cells of the patient. Thirdly, cells obtained
from patients with lipid storage disease and grown in tissue
culture show the same proportional enzyme deficiency as
that which occurs in the organs of the patient from whom
the cells were derived. The acquisition of this information
now permits the accurate diagnosis of individuals afflicted
with any of these lipid storage diseases[”, identification of
heterozygous carriers of these disorders[’], and the prenatal
detection of fetuses amicted with any of these diseases‘’].
Knowledge of the specific enzymatic defects in the hereditary diseases is mandatory for the development of rational
Angew. Chem. inrernat. Edit. J Vol. I 2 (1973)
1No. I
Table 2. Accumulating sphingolipids and missing enzymes in sphingolipidoses. Cer = ceramide; Glc = glucose;
PChol = phosphorylcholine; Gal = galactose; NAcNA = N-acetylneuraminic acid ; NAcGal = N-acetylgalactosamine; Fuc = fucose; NAcGlc = N-acetylglucosamine. The position ofnormal degradation is indicated.
Major sphingolipid accumulated
Enzyme defect
ceramide glucoside
(gl ucocerebroside)
ceramide galactoside
ceramide galactose3-sulfate (sulfatide)
Ceramide lactoside
ceramide lactoside
ceramide trihexoside
ganglioside G,,
hexosaminidase A
Tay-Sachs variant
globoside (plus
ganglioside GM2)
total hexosaminidase
ganglioside G,,,
therapeutic measures for the amelioration of these disorders.
Wherever feasible, due consideration will be given to
potentially useful corrective procedures for the treatment
of patients who are not identified prenatally or where the
parents have elected to continue the pregnancy despite
the identification of an affected fetus.
2.2. Gaucher’s Disease
Gaucher’s disease is the first sphingolipodystrophy in
which the nature of the metabolic derangement was
conclusively demonstrated. A number of important concepts were established in the course of studies on Gaucher’s
disease which have been applied to all other lipid storage
diseases. A brief description of historical developments will
be presented since the discovery of the etiology of many
other inherited disorders followed these precepts.
The lipid which accumulates in the tissues of patients with
Gaucher’s disease is glucocerebroside (Table 2, row 1).
The hexose portion of glucocerebroside is to be contrasted
with that in galactocerebroside, the predominant glycolipid
of the myelin sheath ofnerves (Table 2, row 3).This difference
gave rise to several hypotheses regarding the etiology of
Gaucher’s disease which included :
1)an abnormality of carbohydrate (galactose) metabolism ;
2) an overproduction of glucocerebroside in tissues ; and
3) a deficiency of a catabolic enzyme required for the
degradation of glucocerebroside.
Angew. Chem. internat. Edit. / Vol. 12 (1973) j No. I
The first two hypotheses were shown to be untenable in
studies by Thannhauser[’] and by Trans and Brady[”I,
respectively. The nature of the metabolic defect in
Gaucher’s disease was established by the following series
of experiments. Glucocerebroside was synthesized with 14C
in the D-glucose portion of the molecule[”’. With this
labeled material as substrate, an enzyme was demonstrated
in all of the tissues of the body which catalyzes the hydrolysis of glucocerebroside.
+ H, 0
+ ceramide
This enzyme was found in high activity in the spleen and
was partially purified from this tissue. Optimal incubation
conditions, kinetics, and products of the reaction were
determined. The activity of the glucocerebrosidase was
then measured in samples of human spleen tissue obtained
at operation from a control series of patients and from
patients with Gaucher’s disease. The mean level of glucocerebrosidase activity in the specimens from patients with
the “adult” form Gaucher’s disease was 15% of that found
in the controls[”- l5).Specimens of spleen tissue obtained
from patients with the more rapidly progressing “infantile”
form have extremely little if any glucocerebrosidaseactivity.
The glucocerebroside which accumulates in the spleen,
liver, and bone marrow is derived from lipids in the membranes of senescent red and white blood cells. The principal
neutral glycolipid of leukocytes is ceramide lactoside
(Table 2, row 5). A second important source of glucocerebroside in peripheral tissues is globoside (Table 2, row 8),
the principal glycolipid of red blood cells. These substances
are degraded enzymatically in a stepwise fashion to cer3
amide-glucoside (glucocerebroside) which accumulates in
patients with Gaucher's disease because of the deficiency
of the requisite P-glucosidase. Glucocerebroside also
accumulates in the nerve cells of patients with the infantile
form of Gaucher's disease and probably arises from the
turnover of gangliosides (Table 2, rows 7 and 9).
The diagnosis of Gaucher's disease can be established by
measuring glucocerebrosidase activity with '4C-labeled
glucocerebroside in samples of tissues obtained by biopsy
or in leukocytes obtained from small quantities of venous
blood['61. Leukocyte preparations and cultured skin
fibroblasts are also useful for the detection of Gaucher
The fluorogenic artificial substrate 4methylumbelliferyl P-D-glucoside (Fig. 2) has recently been
used for the detection of homozygotes and heterozygote~['~,'~J.
+ H,O
C H3
+ "
c H3
Fig. 2. 4-Methylumbelliferyl P-D-glucopyranoside, an artificial substrate for measuring P-glucosidase activity. The resulting 4-methylumbelliferone fluoresces.
Diagnostic enzyme assays are of maximum utility when
applied to the monitoring of pregnancies at risk for heritable
metabolic diseases. At the present time the procedure of
choice is the determination of activity in extracts of cultured
fetal cells obtained by amniocentesis (Fig. 3). This technique
may be used for the identification of fetuses which are
heterozygous['] and homozygous['gl for Gaucher's disease.
It is anticipated that eventually artificial substrates will
become useful for prenatal diagnosis of Gaucher's disease.
2.3. Niemann-Pick Disease
Klenk was the first to identify sphingomyelin as the accumulating lipid in patients with Niemann-Pick disease["!
Once again, the deficiency of a catabolic enzyme was shown
by metabolic studies performed with sphingomyelin labeled
in the choline portion of the molecule[211.The mean level
of sphingomyelinase activity in liver preparations from
Niemann-Pick patients was 7% of that of control series["].
Erythrocyte stroma is again a likely source of the accumulating sphingomyelin, since, next to lecithin and phosphatidyl ethanolamine, it is the predominant phospholipid
of the red cell membrane. Sphingomyelin is also a major
lipid constituent of all subcellular elements and the plasma
membrane of cells ; therefore, it seems likely that sphingomyelin could arise as a consequence of turnover of cellular
elements in most if not all tissues.
The diagnosis of Niemann-Pick disease may be confirmed
by determining sphingomyelinase activity in tissue biopsies,
sonicated leukocytes['61, or extracts of cultured skin
fibroblasts[23].An artificial chromogenic substrate, 2-Nacylamido-4-nitrophenylphosphorylcholine,
for measuring
sphingomyelinase activity has recently been proposedlZ4]
(Fig. 4). It must be determined whether this compound
will provide an accurate indication of sphingomyelinase
activity in various cells and tissues. Satisfactory assay
procedures have recently been developed for the detection
of heterozygous carriers of Niemann-Pick disease['. 71.
The prenatal diagnosis of fetuses afflicted with NiemannPick disease is now a proven procedure[251
kO-(CHz)" -CH3
Centrifugation of
concentrate cells
Tissue culture
1 4 - 5 weeks1
Fig. 3. Prenatal detection of genetic abnormalities.
60- (CHdn-CH3
Fig. 4. 2-N-Acylamido-4-nitrophenylphosphorylcholine,
an artificial
substrate for measuring sphingomyelinase activity. The resulting 2 - N acyfamido-4-nitrophenoxideion is yellowish orange.
2.4. Globoid Leukodystrophy :Krabbe's Disease
The metabolic defect in patients with this disorder is a
deficiency of a P-galactosidase which catalyzes the hydrolysis of galactocerebroside (Table 2, row 3)[261.The accuAngew. Chem. internat. Edit. / Val. 12 (1973)
/ NO. I
mulating galactocerebroside is presumed to arise from the
turnover and restructuring of myelin during the development of the nervous system. Patients with this disorder
may be diagnosed by determining galactocerebrosidase
activity in circulating leukocytes and cultured skin fibroblasts[*’! Heterozygotes are best demonstrated through
similar assays with serum samples. The prenatal detection
of a fetus afflicted with Krabbe’s disease has recently been
2.5. Metachromatic Leukodystrophy (MLD)
Sulfatide (Table 2, row 4) accumulates in various tissues of
patients with this disease. The biochemical defect in MLD
is a deficiency of the enzyme which catalyzes the hydrolysis
of s~lfatide[’~,
301. The enzyme is generally designated as
arylsulfatase A because its catalytic activity is frequently
determined using arylsulfate esters as substrate (Fig. 5).
Patients and heterozygous carriers may be detected by
determining arylsulfatase A activity in circulating leukoc y t e ~ [ ~331’ - and extracts of cultured skin fibroblasts[341.
It is anticipated that this assay procedure will be useful for
the prenatal detection of fetuses with MLD.
mulating ceramide trihexoside is globoside (Table 2, row 8)
from senescent erythrocytes.
Enzymatic assay with biopsy specimens of small intestinal
mucosa is a satisfactory procedure for diagnosing patients
A more convenient procedure has
and heterozygote~[~~!
recently been developed in which artificial chromogenic
or fluorogenic a-galactopyranosides are used as substrate~[~’].Refined methods have been developed for
measuring glycolipids in urine sediment and this procedure
is a useful adjunct both for identifying patients with
Fabry’s disease as well as other sphingolipid~ses[~~!
prenatal detection of Fabry’s disease is now an established
2.8. Tay-Sack Disease
This disease occurs in approximately one in 6000 births in
infants of Ashkenazic Jewish parentage. The disorder has
been reported in many other groups, but the frequency in
non-Jews is on the order of one-hundredth of that in the
Jewish race. Histological examination of the brain reveals
swollen neuronal cells in various stages of degeneration,
and many of them contain concentrically-layered electrondense “membranous cytoplasmic bodies” (MCB’s) (Fig. 6).
The MCBs are comprised of protein, cholesterol, phospholipid, and Tay-Sachs ganglioside (GHZ)
(Table 2, row 7),
the pathognomonic substance which accumulates in the
brain of patients with this disorder. Another sphingolipid,
Fig. 5. 2-Hydroxy-5-nitrophenyl sulfate, an artificial substrate for
measuring arylsulfatase activity. The sulfate and 4-nitropyrocatechol
are yellow, the anion (pH = 11-12) is red.
2.6. Ceramide Lactoside Lipidoses
A patient has recently been described in which ceramide
lactoside (Table 2, row 5 ) is the principal accumulating
lipid. The metabolic defect is a deficiency of the P-galactosi
dase which catalyzes the hydrolytic cleavage of the molecule of galactose from ceramide l a c t o ~ i d e ‘ ~For
~ ~ .the
accurate identification of homozygotes and heterozygotesr361the use of galactose-labeled ceramide l a c t o ~ i d e ‘ ~ ~ ]
is mandatory at the present time.
2.7. Fabry’s Disease
This condition is a lipid storage disease in which ceramide
trihexoside (Table 2, row 6) accumulates in various tissues
of these patients, especially in the kidney. The enzymatic
defect is a deficiency of ceramide trihexoside-a-galacto~idase[~*!
Highly purified enzyme has been obtained from
small intestinal tissue[391.The principal source of accuAngew. Chem. internat. Edit. 1 Vol. I 2 (19731 1 N o . I
Fig. 6 . Membranous cytoplasmic bodies (MCB) in a nerve cell from a
patient with Tay-Sachs disease [43]. N =nucleus; NP=nucleolus;
PL =plasma membrane.
asialo-Tay-Sachs ganglioside[*] (ceramide-glucose-galactose-N-acetylgalactosamine) is also increased in the brain
in these infants, but to only one-fifth of the extent of G,,.
The accumulating lipids are primarily confined to the
central nervous system with the notable exception that the
neurons of the intestine are frequently involved.
The discovery of the specific enzymatic defect in Tay-Sachs
disease was hindered by the difficulties encountered in
preparing appropriately labeled G,, for metabolic studies.
This molecule has yet to be synthesized by organic chemists.
In time, GM2labeled with ’H in the N-acetylneuraminic
acid moiety was prepared by a combination of biosynthesis
in ciuo and selective enzymatic degradation of the mixed
radioactive gangliosides obtained in this
labeled with I4C in the N-acetylgalactosaminyl moiety
was also prepared through enzymatic synthesis in ~ i t r o [ ~ ’ ] .
Using thesespecifically labeled substances, it was shown that
the catabolism of G,, can be initiated either by the cleavage
of the molecule of N-acetylneuraminic
or the
molecule of N-acetylgalact~samine~~~~.
activity is normal in tissues obtained from patients with
Tay-Sachs disease[481whereas there is a drastic attenuation
of G , , - h e ~ o s a m i n i d a s e ~ ~These
~ - ~ ~ findings
support the
concept that the hexosaminidase isozyme A which was
shown by artificial substrates to be lacking in patients
with Tay-Sachs disease[”] is involved in the catabolism
of GM2.The complete elucidation of the pathogenesis of
Tay-Sachs disease is complicated by the fact that these
two pathways exist for the degradation of G,, in normal
human tissues and that only one ofthem, the hexosaminidase
route, is disturbed in patients with Tay-Sachs diseaseE49. S O , 521
In the tissues of patients with a second or “0variant” form
of Tay-Sachs disease there is a decrease of total hexosaminidase
This disorder is confined mainly to
patients of non-Jewish extraction. In addition to the
accumulation of G,, in the brains of these patients,
globoside accumulates in peripheral organs (Table 2,
row 8).
There is also a third category of patients with Tay-Sachs
disease called the “AB-variant” in which hexosaminidase
isozymes A and B are both present when assayed with
artificial substrates ; however, the catabolism of G,, is
impaired in these patient^"^! Nevertheless, with the exception of the “AB-variant,” the diagnosis of Tay-Sachs
homozygotes and heterozygotes is readily accomplished
through hexosaminidase assays using serum samples[s41.
The prenatal detection of fetuses afflicted with Tay-Sachs
disease has been reported[”, “I.
2.9. Generalized ( G M ~Gangliosidosis
The principal substance which accumulates in the nervous
system of patients with this disorder is ganglioside G,,
(Table 2, row 9). This condition is therefore frequently
referred to as G,, -gangliosidosis. Along with some accumulation of G,,, a keratan sulfate-like mucopolysaccharide
is increased in the peripheral tissues of patients with this
disorder. The disease is caused by a deficiency of G,,-
N or U-Acylated neuraminic acids are also called sialic acids.
ganglioside-0-galactosidase which catalyzes the hydrolysis
of the terminal molecule of galactose from monosialyltetrahexosyl ganglioside[’’! Patients with this disorder have
a decrease of total tissue P-galactosidase activity as measured with artificial substrates.
Due to this very drastic lowering of galactosidase, it might
be anticipated that other substances having a terminal
molecule of galactose such as galactocerebroside (Table 2,
row 3) or cerarnide lactoside (Table 2, row 5) might accumulate along with GMl.This has not been observed; in
fact, the activity of the enzymes which catalyze the hydrolysis of galactocerebroside and ceramide lactoside are
actually increased 4- to 5-fold in the tissue of these patient~[’~!Nonetheless, artificial galactopyranoside substrates are very useful for the diagnosis of homozygotes,
for identifying heterozyg~tes[’~~,
and for the prenatal
detection of G,,-gangIiosidosis[“]. The accumulating G,,
may arise from the turnover of plasma membrane components of various cells since gangliosides are highly
concentrated in these structuresEfi1,
2.10. Fucosidosis
A discussion of lipid storage diseases should include some
recent observations on a small group of patients with a
disorder called f u c o s i d ~ s i s ~The
~ ~ enzymatic
defect is a
complete deficiency of a-L-fucosidase in the tissues of these
patients[64! The natural substrate(s) of this enzyme is not
well established ; however, it is well known that intestinal
tissue, red blood cells, and certain other tissues contain
fucoglycolipids with H, Lea, and Leb isoantigenic activityL6’].In accordance with the anticipated pathophysiology
of this disorder, a very large increase in pentahexosylfucoglycolipid has been observed in liver tissue obtained
from a patient with fucosidosis[“]. It is presumed that the
specific metabolic defect in this disease will soon be conclusively demonstrated and that it will be a deficiency of
a fucosidase involved in the catabolism of fucolipids
(Table 2, row 10).
3. Treatment
3.1. Gaucher’s Disease
A number of potential forms of therapy have been envisaged
for the correction of inherited metabolic diseasesEfi7.
but so far only a modest amount of direct experimentation
has been carried out. Patients with Gaucher’s disease often
require supportive therapy such as vitamins, supplemental
iron or liver extract for the anemia, and splenectomy
because of the development of hemorrhagic tendencies.
Aside from these palliative measures, the principal concept
for the therapy of Gaucher’s disease is the hope of devising
a procedure for restoring enzyme activity in the tissues of
these patients (Table 3). Since spleen has high glucocerebrosidase activity, transplantation of this organ has
long been considered as a therapeutic procedure for these
patientsEfi7].Techniques have been developed for this
Angew. Chem. internnt. Edit. / Vol. 12 (1973) ,iN o . I
operation and it has recently been performed on a patient
with Gaucher’s disease‘69! The total plasma lipid hexose
level decreased for a short period of time post-operatively
in this patient ;however, there was only a slight diminution
in circulating glucocerebroside. The patient expired three
months after the operation. It must be concluded from this
important study that spleen transplantation is still a very
hazardous undertaking and the efficacy of the procedure
for the treatment of patients with Gaucher’s disease is very
much in doubt.
Table 3. Therapeutic possibilities
1. Enzyme replacement
1.1. Organ transplantation
2. Parenteral administration of purified enzymes
2.1. Animal sources
2.2. Human preparations
2.3. Encapsulated enzyme in biodegradable microspherules
3. Percolation of blood over stably bound enzyme
4. Administration of DNA
4.1. Transducing viruses
5. Anti-enzyme antibodies
6. Hybridization and replacement of patient’s cells
7. Administration of metabolic cooperativity material
The therapeutic approach which is receiving the most
serious consideration at the present time is the possibility
of replacing the missing enzyme by parenteral administration of purified glucocerebrosidase. The enzyme has been
partially purified from human spleen” ‘I, and a more highly
enriched preparation has been obtained from beef spleen
tissue[70! Glucocerebrosidase has recently been obtained
in a high degree of purity from human placental tissuel7*!
We prefer to use enzyme isolated from human sources
because of the expectation that such preparations will be
the least likely to cause an untoward immunological reaction when injected into patients. Considerable experimental evidence indicates that exogenously administered
enzymes will be taken up by the cells of the reticuloendothelial system[721,a highly desirable situation for attempting
to treat patients with Gaucher’s disease. One wonders if
the concentration of exogenous enzyme would be increased
in these cells if the enzyme were encapsulated in a biodegradable “ l i p ~ s o m e ” [ The
~ ~ ~ rationale
of parenteral enzyme
therapy receives some support from investigations in
which a-glucosidase was administered to mice and to
patients with glycogen storage disease. Liver glycogen fell
in the experimental
and in two patients with
g l y c o g e n o s i ~761.
However, a negative report of an essentially similar
therapeutic attempt has appeared[77! All of the preceding
studies were performed with very crude glucosidase
preparations obtained from the fungus Aspergillus niger
and there was a wide variation in the frequency and quantity of enzyme injected. It is therefore virtually impossible
to draw relevant conclusions regarding the effectiveness
of this therapeutic approach from these published reports.
Enzyme replacement procedures may eventually become
useful for reducing the level of the accumulated lipid in
peripheral organs and tissues, but it seems doubtful
Angew. Chem. internat. Edit. 1 Vol. 12 (1973) f N o . I
whether a sufficient quantity of parenterally administered
enzyme would cross the blood-brain barrier to be an
effective form of treatment for patients with central nervous
system difficulties. Alternative procedures have been contemplated for increasing the level of enzymatic activity in
the brain and all of the current concepts seem potentially
quite dangerous. One technique which may be examined
for its clinical usefulness is the temperary opening of the
blood-brain barrier by the intracarotid injection of a
hypertonic solution of urea or other solute[78! Much
refinement and additional experimentation must be carried
out prior to clinical attempts with this procedure since in
its present form it often causes a variable degree of paralysis
in experimental animals.
Another potential form of therapy which may still be
remote, but has recently been brought a little bit closer to
the experimental stage is the long-sought procedure for
transducing human cells with a virus carrying the genetic
message for a missing enzyme-genetic engineeri~~g”~].
Two major obstacles must be overcome before this approach
can be considered appropriate for human trial. The first of
these is the enormous number of infectious virus particles
which must be administered in order to obtain a stably
transduced cell. At the present time, this ratio ranges from
loo0 to 10000 virus particles per cell. The viremia and
viral encephalitis which must necessarily accompany such
a therapeutic attempt are clearly unsupportable. It may
be hoped that procedures will be discovered which will
drastically reduce the presently required high multiplicity
of infectious particles ; for example, development of a technique similar to the use of heat-inactivated Sendai virus
which has been so helpful for preparing hybridized cells.
Secondly, the viruses presently available for cell transduction are primarily of the h-phage group which normally
infect and extract DNA from E. coli. As far as known, this
microorganism does not contain any of the sphingolipid
hydrolases, and therefore another source of the replacement cistron must be sought. One conceivable source may
be H . ciferii, a yeast which is rich in sphingolipids. It is not
known whether an appropriate virus can be obtained for
transduction experiments with such cells.
An additional concept regarding potential forms of therapy
of lipid storage diseases should be borne in mind. It has
been shown in a number of instances that anti-enzyme
antibodies cause an enhancement of enzymatic activity
under certain conditions[80- 831. This is potentially an
important finding since the first two of these reports deal
with the activation of mutant enzymes obtained from
E. coli which are catalytically inactive in the absence of the
appropriate antibody. When antibody was added to the
assay mixtures, the level of enzymatic activity approached
that of the wild type nonmutated enzyme. The potential
importance of this observation is underscored by the fact
that there are proteins in the tissues of patients with
several genetic diseases which cross-react with antibodies
directed towards the normal human e r ~ z y m e ss51.
[ ~ ~It~ is
therefore conceivable that mutated human enzymes with
low catalytic efficiency might be activated through administration of an appropriate antibody. This novel concept also requires extensive additional experimentation
before attempting to exploit this phenomenon for the
treatment of human diseases, since the possibility of
induced amyloid~sis[*~’
must be rigidly excluded.
3.2. Niemann-Pick Disease
Again one must consider the possibifity of exogenous administration of the requisite enzyme. Sufficient quantities
of purified human sphingomyelinase have not been available for therapeutic trials. Perhaps in time it will be possible
to consider liver transplantation for the amelioration of
this disease. Alternatively, it may be worthwhile exploring
the possibility of administering sphingomyefinaseprepared
from other than human sources, such as beef liver or even
a suitably purified enzyme from bacteriarB7!However, an
additional precaution must always be kept in mind when
considering enzyme replacement therapy for NiemannPick disease, namely the ubiquitousness of sphingomyelin.
It is conceivable that the parenteral administration of
sphingomyelinase might cause hemolysis of erythrocytes
and general cellular disintegration with serious sequelae.
In order to preclude such untoward effects, sphingomyelinase may well be an ideal enzyme for administration in
biodegradable microspherules.
3.3. Krabbe’s Disease
Enzyme replacement therapy is high on the list of possible
restorative procedures although again the possibly limited
access ofa parenterally administered enzyme to the nervous
system may preclude its effectiveness.The investigation of
therapeutically distinct approaches in this disease has one
particular advantage which is the well documented observation that certain strains of dogs are affected with a
form of globoid cell leukodystrophy which resembles the
disorder in humans in many respects[8B! A n unusual
opportunity is thereby afforded for examining potential
remedial procedures with this animal model. The principal
limitation at present is the lack of sufficient quantities of
highly purified galactocerebrosidase for these investigations.
Hopefully this restriction will soon be surmounted.
3.4. Metachromatic Leukodystrophy (MLD)
There have been two noteworthy attempts of enzyme
replacement therapy for MLD. The first was carried out
by Austin in 1967, when he infused a relatively crude preparation of human urinary arylsulfatase intrathecally into
a patient with MLD‘”’. The patient had a severe pyrogenic
reaction and there was no evidence of clinical improvement. In another study, a partially purified preparation of
beef brain arylsnlfatase A was administered intravenously
and intrathe~ally[~~*.
The patient became febrile and again
no clinical improvement was observed. Enzymatic activity
was detected in the liver following intravenous infusion,
but no enzyme was found in the brain.
These studies are very discouraging from the standpoint of
enzyme replacement therapy. However, a glimmer of
encouragement may be derived from the recent report that
the quantity of sulfatide in cultured fibroblasts derived
from a patient with MLD decreased when crude urinary
arylsulfatase A was added to the culture medium[911.These
studies re-emphasize the need for devising new concepts
and approaches to try to overcome the enzymatic deficiency
in the nervous system.
3.5. Fabry’s Disease
The administration of moderate quantities of diphenylhydantoin often relieves the pain of the peripheral neuralgia
which rather commonly occurs in teenage patients with
Fabry’s diesease[921.We have obtained homogeneous
preparations of ceramide trihexosidase from human urine
and placenta[931.Replacement trials .with these enzyme
preparations will be undertaken soon.
Another approach which has been suggested for the treatment of patients with Fabry’s disease is the possibility of
kidney transplantation[941. An informative study along
these lines was carried out by PhillippartSg5]who found a
temporary decrease in the level of plasma ceramide trihexoside postoperatively. However, there was a gradual
return of ceramide trihexoside towards the preoperative
level in the plasma after the kidney transplants in spite of
apparently satisfactory function of the grafted kidneys.
Another procedure which has been investigated for the
amelioration of Fabry’s disease is the infusion of fresh
normal human plasma into these patients. It has been
claimed that the circulating cerarnide trihexoside level
decreased as a result of this procedure and that plasma
ceramide trihexosidase activity in the patients increased
to a surprisingIy high valuer961.This report has caused
much concern and more than a little controversy since most
laboratories[97.g81, including the author’s, have been unable
to demonstrate the hydrolysis of galactose-labeledceramide
trihexoside catalyzed by plasma preparations. Therefore,
I feel it is necessary to withhold judgment at this time
regarding the effectivenessof plasma infusion for the treatment of Fabry’s disease.
The possible use of polymer-bound enzymes deserves
consideration at this point. We have veryrecently covalently
coupled ceramide trihexosidase to a polyacrylamide
matrix[991.The rationale for this approach is the desire
to pass whole blood (or plasma) over a column of the stabilized enzyme in the hope that the elevated Ievd of circulating ceramide trihexoside in patients with Fabry’s
disease[’001 will be decreased by this procedure- It is
anticipated that such a lowering of plasma ceramide trihexoside will induce the egress ofthe accumulated glycolipid
from the tissues of these patients. The major obstacle of this
form of therapy is the complicated procedure involved in
providing an absolutely sterile exteriorized arterio-venous
shunt. The dificulties are comparable to those of hemodialysis, a procedure often required for treating patients
with Fabry’s disease. However, an additional constraint
on this procedure for the treatment of Fabry patients is
the fact that the maximal catalytic activity of the enzyme
occurs at pH 5.0; many predictable problems will be
encoiintered if the pH of the blood or plasma is Iowered
to this value.
Angew. Chem. internat. Edit. 1 Vol. I2 (1973) / No. 1
3.6. Tay-Sachs Disease
3.8. Additional Measures
A considerable amount of investigative effort is currently
directed towards enzyme replacement therapy for patients
with the "B-variant" form of Tay-Sachs disease. The missing
enzyme, hexosaminidase A, has been purified to homogeneity from human placenta and urine['0''. An investigation
has been carried out on the distribution of the enzyme
after intravenous infusion in a patient with Tay-Sachs
disease. Enzymatic activity disappears very rapidly from
the blood stream with the kinetic course indicated by the
A potential corrective procedure which is receiving considerabIe attention at the present time is the possibility of
hybridizing some of the patient's cells with other human
cells which contain the cistron for the defective enzyme.
A very interesting experiment along these lines was reported
by Nadler et al. who hybridized human cells from patients
with gala~tosemia"~~!
These individuals are deficient in
the enzyme galactose-I -phosphate uridyl transferase :
Hexosaminidase activity
Some hexosaminidase A activity appears in the liver after
infusion; however, only a very small amount of enzyme
entered the brain. Obviously, much additional investigation must be carried out along these lines.
Since there is considerable hexosaminidase A and B activity
in serum, as measured with artificial substrates, a number
of therapeutic trials may be expected utilizing fresh human
plasma as a source of hexosammidase. There are several
major drawbacks to this approach among which are the
large quantities of plasma which would have to be infused ;
however, this limitation might be circumvented to a degree
by enzyme concentration procedures.
A second, more important drawback, is the complete lack
of evidence that circulating plasma hexosaminidase catalyzes the hydrolysis of G,,, the accumulating lipid.
Thirdly, there is the ever-present doubt whether a sufficient quantity of enzyme can enter the brain from the
blood. Other than altering the permeability of the bloodbrain barrierc7*]which is at best a potentially hazardous
procedure, or genetic engineering[791which is currently
viewed with extreme skepticism, the most logical approach
appears to be to try to devise a procedure to chemically
modify hexosaminidase so that it will pass through the
blood-brain barrier while retaining activity towards the
natural substrate. I suggest that an attempt be made to
chemically modify the enzyme by the covalent attachment
of medium chain length aliphatic amino alcohols to the
protein. I predict that this procedure will be a principal
avenue of investigation for the treatment of Tay-Sachs
disease in the near future.
3.7. G ,
+ uridinediphosphate glucose
+ glucose-I -phosphate
uridinediphosphate galactose
The hybrid cells contained catalytically active enzyme.
These data suggest that the enzymatic defects in the parent
cell lines weredue todifferent point mutations in the genetic
code and that interallelic complementation (correction)
had occurred in the hybrid cells. These experiments open
the possibility of reimplanting cells in patients after hybridization has been carried out in tissue culture.
Two very important precautions must be constantly kept
in mind when contemplaling the therapy of human disease
by this procedure. The first is the danger of producing an
antigenically incompatible cell which would be rapidly
rejected by the recipient. Perhaps this potential difficulty
can be circumvented by proper selection and cloning of
hybridized cells whose complement of antigens is compatible with the recipient. Secondly, and perhaps potentially even more hazardous, is the fact that spontaneous
malignant transformation isnot an infrequent phenomenon
in cultured cells. It is especially likely to occur if repeated
passages of the cells are carried out. Tests to exclude this
untoward reaction are very difficult since they would have
to be performed in experimental animals. The reliability
ofdata obtained in this fashion for extrapolation to humans
would certainly be questionable.
Some very recent reports contain data which permit my
closing this dissertation on a cautious note of optimism.
In 1966, Suback-Sharpe, Biirk, and Pitts reported that baby
hamster kidney cells which were deficient in inosinic acid
pyrophosphorylase (Fig. 7) showed evidence of correction
Concepts relevant to the treatment of patients with this
disorder generally follow those described for Tay-Sachs
disease and the other lipid storage diseases. The P-galactosidase involved has been obtained in a purified form from
mammalian liver tissue[i031.One important advantage of
this enzyme preparation is its high activity toward G,,,
the natural lipid substrate. However, the precautions
discussed in the section on Niemann-Pick disease regarding
enzyme infusion may well apply when considering replacement trials for G,,-gangliosidosis since gangliosides
are important components on the surfaces of many cells.
Angew. Chem. internat. Edit. J Vol. I 2 (1973) J No. I
OH 6 H
Fig. 7. Hypoxanthine and cc-5-phospho-6-ribosyl I-pyrophosphate are
normally transformed into inosinic acid and pyrophosphate by the
action of inosinic acid pyrophosphorylase.
of the metabolic defect if they were grown in the presence
of normal cells or ceIls deficient in a different enzyme[i051.
This observation has been amply confirmed and the restoration phenomenon is called "metabolic cooperativity". It
appears that a "factor" passes between the cells and the
agent responsible for such correction in patients with
mucopolysaccharidoses appears to be a protein["'!
Similar corrective factors have been found in blood plasma
and urine. The latter observations prompted Di Ferrunte
et ul. to undertake therapeutic trials of plasma infusion in
patients with mucopolysaccharide
investigators were encouraged by their results and the
experiments were extended to an examination of the effect
of transfusing leukocytes in a patient with a similar disorder[i08! The patient was said to have benefited by this
procedure and the results were considered to be superior
to those obtained with plasma infusion.
These reports have prompted a spate of clinical investigations along this line and both positive[i0g1and negative"
clinical responses have been obtained. It is my feeling that
this technique clearly warrants extensive investigation at
this time. It is certain that leukocyte transfusion will be
tried in other metabolicdiseases, for example in the sphingolipidoses. The reasonableness of this approach is indicated
by the fact that leukocytes contain glucocerebrosidase["],
galactocerebrosidase[' 'I, and sphingomyelinase[i"l activity. Caution and restraint are required on the part of the
investigator lest he carry this concept too far. For example,
implantation of bone marrow cells has been suggested as
a possible approach for providing a stable, constant
source of cells for patients with immunological deficiencies.
It was concluded in a recent survey that this form of
therapy is a very hazardous procedure at this time["'].
4. Conclusions
I have attempted to provide a precis of current concepts for
the therapy of inherited diseases and to indicate points of
departure for future explorations. Circumspection, imagination, and caution are required in order to mount effective
therapeutic regimes for the treatment of these disorders.
A number of potential approaches are described and
evaluated in this review. Some of these possibilities are
worthy of exploration at this time ;others must await further
innovation and improvement. High on the list of potentially effective procedures are replacement trials with
purified enzymes and the administration of metabolic
cooperativity factors. These approaches appear likely to
be beneficial for a limited number of conditions. Other,
more generally useful, forms of therapy must be developed
for the amelioration of the majority of heritable human
Received: February 17,1972 [A 918 1E]
German version: Angew. Chem. 85,28 (1973)
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