Introduction

Schwann (1839) was the first to use the term metabolic phenomena. He defined metabo-
lism as "the sum total of chemical transformations which take place in living cells or in
the surrounding internal environment through the activity of these cells." The term, or its
vernacular equivalents, is now universally accepted. Discoveries in the field of biochem-
istry have led to a more precise definition of the concept while retaining the overall
meaning of the word. A brief outline of the evolution of this concept is essential for un-
derstanding the pathophysiology of metabolic diseases.
Metabolism is confined to living organisms. The cell, as the smallest living unit, is the
site of continuous metabolic activity. This consists of the synthesis (anabolism) and
degradation (catabolism) of living constituents, as well as processes providing energy for
various bodily functions. Most of these chemical reactions would run too slowly to be ef-
fective, if not accelerated by the catalytic action of enzymes. Over 1000 of these biologi-
cal catalysts have been identified to date and more are discovered continuously. The en-
zymes have the structure of proteins of relatively high molecular weight. They are highly
specific both antigenically and in their catalytic activities.
Enzymes, like other proteins, undergo continuous synthesis and degradation. Their
synthesis is controlled by the genetic code enshrined in the structure of DNA, which
forms the template for the amino acid sequence of the proteins.
The structure of proteins may be considered on several levels: the amino acid sequence
of the polypeptide chain (primary structure), the helical or sheetlike configuration of the
chain (secondary structure), the three-dimensional spatial arrangement of the chain (ter-
tiary structure), and finally the linkage of several chains that form the subunits of a bio-
logically active protein (quaternary structure). Even minor changes in the primary struc-
ture, such as the substitution of a single amino acid, may have far-reaching consequences
for the tertiary and quaternary structures, and thus for the functional activity of the en-
zyme.
The enzyme systems are not uniformly distributed within the cells. Glycolytic enzymes
are localized in the cytosol; those of the respiratory chain form part of the mitochondria,
4 Introduction

while hydrolytic enzymes operate in the lysosomes. The enzymes form an integral part of
the structures of the subcellular organelles; hence, alterations in enzyme structure may be
reflected in morphological changes within the cell. Cellular structure and metabolism are
so closely coordinated that certain specific structural changes may hint at the underlying
metabolic error. While care must be taken not to confuse artifacts with genuine lesions,
immunohistochemistry has revealed generally good preservation of molecular structures
in routine histological preparations. The lysosomal and peroxisomal apparatus is well vi-
sualized by electron microscopy, which has given valuable insight into the pathogenetic
mechanisms operating in some metabolic disorders.
Every disease may be accompanied by metabolic disturbances. Environmental factors
(e.g., malnutrition, vitamin deficiencies, and poisons) and auxiliary mechanisms (e.g.,
disorders of absorption, transport, and excretion) must be distinguished from primary
metabolic disorders due to the defective activity of one or more enzymes. The latter are
discussed in this volume, inasmuch as they affect the central nervous system (CNS) and
the peripheral nervous system. Most of these diseases are rare; some, exceedingly so.

Fundamentals of Metabolic Diseases

The maintenance of all vital functions depends on the integrity of metabolic

processes. Atrophy and necrosis result from global impairment of metabolic functions,
due to imbalance, or arrest, of the processes of synthesis and degradation. Metabolic
diseases do not, as a rule, involve such global processes, but depend on the impair-
ment of synthesis or breakdown of specific substances in the living cell. The intracel-
lular deposition of abnormal substances, recognizable by routine histological methods
and roughly identifiable chemically, led to the concept of storage disease.
The term storage disease was first applied by von Gierke (1929) to glycogenosis.
The concept of true storage diseases was confined to disorders in which retention of
intermediate products of cellular metabolism was an essential and irreversible feature
(Siegmund, 1938; Giampalmo, 1951).
It is erroneous to consider storage to be a primary pathogenetic mechanism, how-
ever impressive it may be to the morphologist. Rather, it is an epiphenomenon of the
underlying metabolic defect. Nevertheless, the chemical nature of the stored sub-
stances remains an essential criterion for the classification of the metabolic diseases,
at least for the time being.

Lysosomal Storage Diseases

Lysosomes are organelles characterized by an extraordinary abundance of acid hy-

drolyses. The number of enzymes identified in the lysosomal fractions has increased
considerably over the years. Increased activity of this enzyme is a common finding in
lysosomal diseases in which acid phosphatase can be demonstrated histochemically.
The lysosomes are enveloped in a membrane corresponding in thickness to the
plasma membrane of the cell surface (de Duve, 1969). They contain products of
Lysomal Storage Diseases 5

degradation both in the normal process of catabolism and in pathological storage. In

the latter situation the stored material is found in residual bodies (see below). The
term lysosomal storage disease was introduced by Hers (1964) and is now generally
accepted. The appearances of the lysosomes depend on the nature of the stored mater-
ial, which, in some disorders, may be sufficiently characteristic to allow a morpholog-
ical diagnosis.
The morphological heterogeneity of lysosomes is due to both the nature of the in-
gested material and its means of entry. The presentation of the material differs be-
tween heterophagy (the breakdown of exogenous substances) and autophagy (the
breakdown of cell components). As a rule, heterophagy plays no part in most meta-
bolic diseases.
The lysosomal degradation of a cell's own constituents is part of the normal
turnover of cell components. This does not involve ingestion of materials from outside
and can also be divided into three phases.

1. The sequestration of an area of cytoplasm leads to the formation of an au-

tophagosome, which does not contain lytic enzymes. This involves the formation of a
membrane demarcating the sequestrated portion (Pfeifer, 1987).
2. As the degradation proceeds a secondary lysosome is formed. As the process of
autophagic digestion occurs continuously, one must postulate a cyclical turnover of
lysosomes, in which newly formed ones may play a part.
3. In the process of digestion, all formed and unformed constituents are broken
down into micromolecular fragments. After the completion of lysosomal degradation,
the secondary lysosomes become telolysosomes (de Duve and Wattiaux, 1966). Sec-
ondary lysosomes that retain incompletely digested material are called residual bodies.
They are the morphological substrate of the lysosomal storage diseases, but are also
formed in nonpathological cells.

As autophagy is a continuous process in normal cells, they contain a normal com-

plement of lysosomes. However, as metabolic processes differ in individual cell types,
their residual bodies display qualitative and quantitative differences. Lipofuscin gran-
ules (Fig. 1A) are present in residual bodies in normal neurons. The residual bodies of
oligodendrocytes contain "fingerprint" bodies (Fig. 2). The uniformity of these two
types of residual bodies suggests that they are both autophagic in nature.
By contrast, the astrocytes, which are capable of heterophagy, contain highly pleomor-
phic residual bodies (Fig. 3). The pericytes of cerebral capillaries and the adventitial cells
of arterioles and venules display characteristic globular lipid inclusions (Fig. 1B).
Transient overloading of lysosomes may occur when the uptake of substrate is
greater than the lysosomes can digest. A good example is the accumulation of protein
droplets absorbed by the proximal renal tubules.
Some proteins, such as immunoglobulins and collagen, are relatively resistant to the ac-
tion of lysosomal proteases, and, under conditions of rapid endocytosis, many accumulate
in secondary lysosomes. Intensification of autophagy may lead to a striking increase in the
number of lysosomes. Glucagon stimulates a multiplication of autophagic vacuoles (Deter,
1971). Ionizing radiation also causes increased autophagy (Cerv6s-Navarro, 1964).
6 Introduction

Fig. 1 (A) Lipofuscingranules, x20,000.

Experimental inhibition of lysosomal enzymes leads to a temporary and reversible in-

crease in the number of residual bodies (de Duve, 1983; Henell and Glamann, 1984). A
permanent overload may occur in incomplete digestion, in which each cycle adds to the
residue. This may occur when indigestible substances, such as dextran, find their way
into lysosomes (Roberts et al., 1976; Pfeifer et al., 1984) or when an enzyme is missing,
as in genetic storage diseases. In these situations the time factor must be taken into ac-
count, as a certain length of time is required for the accumulation to become morphologi-
cally appreciable.
Certain basic ultrastructural patterns may be identified (Figs. 4-9). Even if these pat-
terns are not pathognomonic for any particular disease entity, they are at least partially
specific and offer useful diagnostic pointers.

Disorders of Other Organelles

Although lysosomal diseases account for the bulk of metabolic disorders, many dis-
eases are caused by the malfunction of other organelles and deficiencies in their enzymes.
Abnormalities in mitochondrial and peroxisomal functions have attained increasing im-
portance in recent years. The mitochondrial enzymes are predominantly dehydrogenases,
Enzymopathies 7

Fig. 1 Continued.(B) Lipophagosomalresidual bodies, x 12,000.

primarily involved in oxidative phosphorylation. They are therefore responsible for the
normal functioning of the respiratory chain, and deficiencies in these enzymes can lead to
disease processes in which this metabolic cascade is interrupted.
Peroxisomes contain a great variety of enzymes, peroxidases, catalases, and several en-
zymes active in the degradation of very long-chain fatty acids. A deficiency of these en-
zymes can lead to several recently recognized diseases, the hallmark of which is storage
of these fatty acids.

Enzymopathies
In a study of alkaptonuria, Garrod (1908) assumed a congenital absence of an enzyme
and developed the concept of inborn errors of metabolism. Beadle and Tatum (1941)
8 Introduction

Fig. 2 "Fingerprint"bodies, x 125,000.

demonstrated in mutants of bread yeast (Neurospora crassa) that the genetic control of
metabolism operates through the genetic control of enzyme synthesis. This confirmed
Garrod's hypothesis and defined the inborn errors of metabolism as permanent, geneti-
cally conditioned, metabolic disorders caused by enzymatic defects.
The absence or functional insufficiency of a specific enzyme cannot always be re-
duced to a single "missing enzyme." The concept of enzymopathy is wider than that of
a "defective enzyme" or a missing enzyme. In theory, at least, one can distinguish en-
zymopathies with reduced enzyme, activity from those with increased activity. As a
rule, only the former manifest themselves as diseases in which a definite metabolic
function is abolished or inadequate. The formation or breakdown of a specific sub-
stance then becomes impossible or greatly impaired. A gradual reduction of enzyme
activity and disturbances of interaction between enzyme and substrate are also in-
cluded in the concept of enzymopathy. Enzymopathies may be inherited or acquired.
Only the former represent the classical inborn errors of metabolism.
The activity of enzymes may be impaired or enhanced by a variety of factors (e.g.,
nutritional or hormonal). The synthesis may be subject to similar influences. The se-
quence of aminoacids in any specific enzyme is genetically determined and coded in
the nucleic acid sequence of DNA (structural genes). It used to be assumed that a de-
Enzymopathies 9

Fig. 3 Pleomorphicinclusions, x 12,500.

fective gene abolished the synthesis of the corresponding enzyme, which was there-
fore missing (Tatum, 1959). In many cases, however, a minor alteration of the DNA
sequence will result in not a missing, but a modified, enzyme. Some enzymes are
composed of several polypeptide chains, each of which is coded by a different gene.
The integrity of such enzymes depends on the preservation of all relevant genes.
Most enzymopathies are due to mutations in structural genes, coding for specific
enzymes, but other mechanisms may lead to impaired enzyme activity. A progressive
loss of enzymes may be involved in muscular dystrophy, in which a defect in the cell
membrane may lead to leakage of metabolic components from the interior of muscle
cells. This may be compensated for some years by increased enzymatic synthesis of
the lost components, but ultimately this fails, leading to an irreversible loss of tissue.
The uptake of lysosomal enzymes depends on the presence of specific receptors
(Neufeld et al., 1977). Some lysosomal storage diseases may therefore be due to re-
ceptor defects.
Hydrolases, like all other enzymes, are hydrophilic. In vitro, they show little activ-
ity in the degradation of lipophilic substances. This is largely due to the fact that
10 Introduction

Fig. 4 Lamellar inclusions. (A) With concentric membranes, x34,800. (B) With multiple small membra-
nous whorls, x 10,440. (C) With irregular membranes, x52,200. (D) With parallel bilaminar mem-
branes, x 52,200.
Enzymopathies 11

Fig. 5 Concentric lamellar inclusions with a dense center, x38,000.

Fig. 6 Zebra bodies, •

Fig. 7 Multivesicular bodies, x40,000.

Fig. 8 Curvilinear inclusions, X28,000.

Classification of Metabolic Diseases 13

Fig. 9 Elongated bilaminar structures, x 10,000.

lipids, particularly apolar ones (e.g., triglycerides and cholesterol), form aggregates
(micelles) that are impenetrable by the enzymes. The activity of the hydrolases was
explained by the discovery of nonenzymatic protein activators. The first such activa-
tor, necessary for the degradation of sulfatides by arylsulfatase A, was isolated by
Mehl and Jatzkewitz (1964). The activator forms a complex molecule with the lipid,
converting it into a suitable substrate of the enzyme. The absence of appropriate acti-
vators explains the pathogenesis of some metabolic disorders in which storage occurs
despite normal enzyme activity in vitro.

Classification of Metabolic Diseases

In spite of the recent contributions of molecular biology, our knowledge is still too
limited for a classification based on identification of the enzyme. To provide a com-
prehensive classification of metabolic disorders, useful to both the clinician and the
pathologist, a variety of criteria should be adopted.
A classification based on storage phenomena, however, is not universally applica-
ble, as, in some conditions, there is no morphologically demonstrable storage of
metabolites. This applies particularly to disorders in which the retained substrate is
water soluble and extended in the urine. This, as well as the similarity of various stor-
age diseases under light microscopy, renders a purely phenotypic classification system
insufficient.
14 Introduction

Electron microscopy has added a refinement to phenotypic classification when it

became apparent that many stored substances had characteristic ultrastructural pro-
files. The chemical characterization of stored substances led to an improved classifica-
tion of metabolic disorders based on classes of substances. Further investigations,
however, have revealed that substances of different classes may be stored simultane-
ously. This may occur when a defective enzyme is responsible for the degradation of
complex substances consisting of carbohydrates, amino acids, and/or lipids.
This characterization has already been achieved in many metabolic disorders and
has been of great importance in the isolation of subtypes and variants. The identifica-
tion of structural defects in enzymes plays an increasingly important role in the sys-
tematic classification of metabolic disorders. We are still faced with difficulties, how-
ever, as a large number of conditions continue to await identification of the
responsible enzyme.

Genetics of Metabolic Diseases

The concepts of dominance and recessivity were originally defined by Mendel

(1965). Recessive alleles are expressed only in homozygotes. Dominant alleles mask
or suppress the expression of the other (recessive) allele of the same gene. These
genes therefore express themselves in heterozygotes. It may be added that these con-
cepts are not absolute and that a range of expressivity may be formed between the two
extremes. The mechanism of recessive inheritance is generally better understood than
that of dominance. Most enzymopathies are inherited as autosomalmsome, as sex-
linkedmrecessives. The gene expression is subject to a regulatory mechanism.
In all highly developed multicellular eukaryotes the cells undergo differentiation,
leading to a multiplicity of cell types. In the human body there are about 200 different
cell types, each of which contains an identical genome. Each cell synthesis yields be-
tween 10,000 and 20,000 different proteins, many of which are specific to a particular
cell type. In this process only 7 - 1 0 % of the entire genome is expressed in each cell. It
is likely that the remaining 9 0 - 9 3 % of the genes should be inactivated by specific re-
pressor proteins. A regulatory mechanism must therefore exist to control correct gene
expression.
The sequence of events leading to gene expression is now well understood, albeit
with some gaps in our knowledge (Willman, 1993; Latchmann, 1993; Rosenthal,
1994). The human genome contains 100,000 distinct genes, encoding all structural and
functional proteins. Every cell contains two alleles of these "single-copy" genes: one
of maternal origin, the other of paternal origin. These single-copy genes compose only
3 - 5 % of the 3.2 billion nucleotide base pairs (bp) that make up the nuclear human
genome. The vast majority of the genome is composed of noncoding DNA, the func-
tion of which has not been fully elucidated.
Each gene consists of a DNA strand divided into several encoding sequences (ex-
ons) separated by nonencoding ones (introns). Attached to the 5' end of the gene, there
is a promoter, consisting of adenosine and thymidine nucleotides (the "TATA box")
Genetics of Metabolic Diseases 15

and a variety of enhancers. Transcription starts by the action of the enzyme RNA poly-
merase associated with a number of transcription factors. The whole DNA sequence is
transcribed to form the premessenger RNA. The transcript is then processed in several
stages. A nucleotide cap is added to the 5' end while several bases are trimmed from
the 3' end by the action of the RNA clipping enzyme. A polyadenosine tail is added to
the trimmed 3' end by the adenosine adding enzyme (terminal transferase). Spliceo-
somes then excise the introns and join the ends of the exons. Apart from this regular
splicing, "alternative splicing" also occurs, by which the exons are rearranged and
some are omitted. The mechanism of this process is not understood, but it enables
each gene to code for several different, but related, proteins. The processed transcript
becomes the messenger RNA, (mRNA) which leaves the nucleus for the cytoplasm,
where it enters the ribosomes. The mRNA is then translated into proteins, each nu-
cleotide triplet coding for a different amino acid. At the end of this chapter, abbrevia-
tions of the amino acids are listed. Posttranslational modifications may still alter the
end product.
The whole process is regulated by other genes, enzymes, growth factors, inducers
from neighboring cells or tissues, and hormones. In view of the complexity of the
process, it is surprising that faulty end products are comparatively rare. The main
types of genetic aberrations fall into the following groups: (1) point mutations, in
which a substituted nucleotide in a triplet codes for a different amino acid; (2) dele-
tions, in which an exon or part thereof is excised in the process of splicing, sometimes
with the insertion of a foreign sequence; and (3) repeats, in which a nucleotide
triplet is repeated sequentially, forming a long chain. All of these cause the formation
of a missense or nonsense end product, whether it be an enzyme or a structural pro-
tein.
A small quantity of DNA is also present in mitochondria (mtDNA). The mitochon-
drial genome consists of about 16.5 kilobases (kb), arranged in two strands: a heavy
one and a light one. Each strand has its own promoter that initiates transcription. The
mitochondrial genome encodes for 13 structural proteins, two ribosomal RNAs
(rRNAs), and 22 transfer RNAs (tRNAs). Each cell contains multiple mitochondria,
and each mitochondrion includes several copies of its genome.
Mitochondria are inherited maternally, as only the naked nucleus of the spermato-
zoon enters the ovum during fertilization. During cell division mitochondria are dis-
tributed at random. In normal tissues all copies of the mitochondrial genome are iden-
tical (homoplasmy). In most pathological situations both normal and mutant genes
may be present (heteroplasmy). A certain minimum number of mutant genes must be
present in order to be expressed (the threshold effect). The threshold may vary in dif-
ferent tissues according to each tissue's energy requirements. Because of the random
distribution of mitochondria, only some offspring of an affected mother may reach the
threshold and express the pathological phenotype.
Several methods are now available for the localization or identification of abnormal
genes. Naturally occurring variations in DNA sequence (polymorphisms) have been
exploited in the identification of disease-associated genes. Polymorphisms in human
DNA occur predominantly in two forms: (1) restriction fragment-length polymor-
16 Introduction

phisms (RFLPs) and (2) variable-number tandem repeats. RFLPs are variations in
DNA sequence between individuals, which abolish or create new restriction endonu-
clease clearage sites. Using DNA probes that are complementary to a polymorphic re-
gion of DNA, variation in a restriction endonuclease cleavage site in this region in dif-
ferent individuals can be detected by Southern blot analysis.
Polymorphisms in DNA sequence surrounding single-copy genes have no effect on
gene function. However, if these polymorphisms are adjacent to (i.e., "tightly linked"
with) a gene of interest (e.g., a gene for a distinct disease), they are said to be "infor-
mative" and may be used for disease diagnosis. In the study of a family with a genetic
disease, it may be found that a polymorphism always cosegregates with the clinical
manifestation of the disorder. This would imply that the particular polymorphism is
lying near the gene containing the disease mutation. This polymorphism can then be
used as a marker of the disease gene, even though the precise location, structure, se-
quence, and function of the disease gene are unknown. These disease genes, which are
localized by polymorphic markers, can be produced in large amounts by molecular
cloning and subsequently rapidly and easily sequenced. Thus, they can be identified.
Alterations in these genes, (e.g., mutations) can be studied by direct sequencing
techniques of DNA fragments containing the mutations of interest or by other meth-
ods, for example, single-stranded conformation polymorphism analysis, which allows
the detection of polymerase chain reaction-amplified DNA sequences containing sin-
gle point mutations.

Phenotype and Genotype

Numerous diseases considered nosological entities have been shown in the last 20
years to be disease groups, consisting of a number of types clearly separable by clini-
cal, biochemical, or genetic criteria. The pioneering work of Cori and Cori (1952) re-
vealed that the glycogen storage disease of von Gierke was not a single entity, but a
large group of glycogenoses, each of which depends on a different, specific, geneti-
cally determined enzyme defect. This development affected almost all aspects of clini-
cal genetics. It is therefore difficult to deduce the nature of the genotype from the phe-
notype, all the more so if the latter is influenced by environmental factors. A good
example is phenylketonuria (see p. 169), in which mental retardation and other stig-
mata of the disease, caused by a defect in phenylalanine hydroxylase, can be pre-
vented by appropriate dietary treatment.
A single gene may affect more than one phenotypic feature, a situation known as
pleotropism. A mutation in a pleotrophic gene may lead to multiple changes in the
phenotype. On the other hand, an identical or similar phenotype may result from muta-
tions at different gene loci (genocopy or heterogenesis). A similar phenotype may also
arise from a variety of alleles at the same locus (genetic heterogeneity).
When the primary enzymatic defect is unknown, it may be difficult to decide be-
tween variants of a single entity and genetically distinct entities. Hybridization of
some somatic cells in tissue cultures may help in solving these problems (Ruddle et
al., 1982).
Abbreviations of Amino Acids 17

Abbreviations of Amino Acids

The common abbreviations of amino acids use either three-letter or single-letter code.

Ala, or A Alanine Leu, or L Leucine

Arg, or R Arginine Lys, or K Lysine
Asn, or N Asparagine Met, or M Methionine
Asp, or D Aspartic acid Phe, or F Phenylalanine
Cys, or C Cysteine Pro, or P Proline
Gln, or Q Glutamine Ser, or S Serine
Glu, or E Glutamic acid Thr, or T Threonine
Gly, or G Glycine Trp, or W Tryptophan
His, or H Histidine Tyr, or Y Tyrosine
Ile, or I Isoleucine Val, or V Valine