A Clinical Guide to Inherited Metabolic Diseases / Edition 3

1

General principles

Introduction

In his 1908 address to the Royal College of Physicians of London, Sir Archibald Garrod (1857-1936) coined the expression inborn error of metabolism to describe a group of disorders - alkaptonuria, benign pentosuria, albinism, and cystinuria - which ". . . apparently result from failure of some step or other in the series of chemical changes which constitute metabolism".¹ He noted that each was present at birth, persisted throughout life, was relatively benign and not significantly affected by treatment; and that each was transmitted as a recessive trait within families in a way predictable by Mendel's laws of inheritance. In fact, alkaptonuria was the first example of Mendelian recessive inheritance to be recognized as such in humans. Garrod concluded, from the results of experiments on the effects of feeding phenylalanine and various putative intermediates of phenylalanine metabolism on homogentisic acid excretion by patients with the condition, that homogentisic acid is an intermediate in the normal metabolism of phenylalanine and tyrosine. Moreover, he observed that the specific defect was in the oxidation of homogentisic acid. La Du subsequently confirmed this 50 years later by demonstrating profound deficiency of homogentisic acid oxidase in a biopsy specimen of liver from a patient with alkaptonuria.²

Following F⊘lling's discovery of phenylketonuria (PKU) in 1934, Garrod's concept underwent a major change, particularly with respect to its relationship with disease. Like alkaptonuria, PKU was shown to be caused by a recessively inherited point defect in metabolism, in the conversion of phenylalanine to tyrosine in the liver. However, unlike Garrod's original four inborn errors of metabolism, PKU was far from benign - it was associated with a particularly severe form of mental retardation. Moreover, although the underlying metabolic defect was 'inborn' and persisted throughout life, the associated mental retardation could be prevented by treatment with dietary phenylalanine restriction.

Harry Harris (1919-1994) applied the technique of starch gel electrophoresis, described originally by Smithies in 1955, to the demonstration of a large number of protein polymorphisms in humans, confirming the vast biochemical genetic diversity predicted by Garrod. He went on to show how the principle of one gene-one polypeptide chain applied to Garrod's inborn errors of metabolism, which he reviewed in some detail in his highly successful book, The Principles of Human Biochemical Genetics³, published originally in 1970.

The discovery of PKU sparked the search for other clinically significant inborn errors of metabolism. The most recent edition of The Metabolic and Molecular Bases of Inherited Disease⁴, edited by Charles Scriver and his colleagues, reports that the number of diseases in humans known to be attributable to inherited point defects in metabolism now exceeds 500. While the diseases are individually rare, they collectively account for a significant proportion of illness, particularly in children. They present clinically in a wide variety of ways, involving virtually any organ or tissue of the body, and accurate diagnosis is important both for treatment and for the prevention of disease in other family members.

While inborn errors of metabolism are a well-known cause of disease in children, their contribution to disease in adults is less well appreciated. This may be due in part to the lethal nature of many of the diseases, which often cause death before the patient reaches adulthood. However, owing primarily to advances in diagnostic technology, especially molecular genetics, a rapidly growing list of inherited metabolic diseases presenting in adulthood is emerging. Some of these, such as Scheie disease (MPS IS), are milder variants of diseases caused by the same enzyme deficiency that commonly causes death in childhood. Others, such as classical hemochromatosis, almost never present in childhood, though the disease in adults may be severe.

The purpose of this book is to provide a framework of principles to help clinicians recognize when an illness might be caused by an inborn error of metabolism. It presents a problem-oriented clinical approach to determining the type of metabolic defect involved and what investigation is needed to establish a specific diagnosis.

Some general metabolic concepts

Metabolism is the sum total of all the chemical reactions constituting the continuing process of breakdown and renewal of the tissues of the body. Enzymes play an indispensable role in facilitating the process by serving as catalysts in the conversion of one chemical (metabolite) to another, often extracting the energy required for the reaction from a suitable high-energy source, such as ATP. All enzymes have at least two types of physico-chemical domains: one or more substrate-binding domains, and at least one catalytic domain. Mutations might affect enzyme activity by affecting the steady-state amount of enzyme protein because of a defect in enzyme production or as a result of abnormally rapid breakdown of the mutant protein. Deletions and insertions causing shifts in the reading frame, mutations to premature stop codons, and mutations affecting the processing of the primary RNA transcript all affect enzyme activity by decreasing the production of active enzyme protein. In contrast, studies on the turnover of mutant enzyme proteins have shown that many single amino acid substitutions resulting from single base substitutions cause deficiency of enzyme activity by causing abnormal folding of the nascent enzyme polypeptide which in turn causes aggregation and premature destruction of the polypeptide before it leaves the endoplasmic reticulum. This discovery is of signal importance to the development of new and specific treatments for diseases caused by inborn errors of metabolism because the stability of many mutant enzyme proteins, and therefore the activity of the enzymes, is enhanced by exposure to 'chemical chaperones'. These are relatively simple and inexpensive chemicals, in some cases substrate analogues, that bind to the mutant enzyme protein, preventing premature degradation. Mutations might also impair the activity of the enzyme without affecting the amount of enzyme protein by specifically impairing the catalytic properties of the protein. Of all the possible mechanisms of enzyme deficiency caused by mutation, this is the most uncommon.

The rapid transport of metabolites across cellular and subcellular membranes is facilitated in many cases by specific transport proteins that function like enzymes. This means that the process is susceptible to genetic mutations affecting the amount or function of the transporter in exactly the same way that mutation affects the activities of enzymes, and with similar consequences.

The coding sequences of most structural genes are comprised of at least a few thousand nucleotides, and the potential for mutation-generated variations in nucleotide sequence is vast. In the same way, the effects of mutation also vary tremendously. At one extreme, some mutations may totally disrupt the production of any gene product, resulting in severe disease. By contrast, other mutations might have no effect whatsoever apart from a functionally silent change in the nucleotide sequence of the gene. The relationship between genotype and disease phenotype is complex. Severe mutations, such as deletions or insertions, are generally associated with clinically severe disease, and the disease phenotype among different affected individuals tends to be similar. Structurally more subtle mutations, such as those resulting in single amino acid substitutions, are often associated with milder disease phenotypes. Moreover, the disease phenotype often varies markedly between different affected individuals, even within the same family, a reminder that the expression of any genetic information, including disease-causing mutations, is influenced by other genes (gene-gene interactions) and by environmental factors (gene-environment interactions).

Figure 1.1 The primary consequences of inborn errors of metabolism. The figure shows diagrammatically the various possible mutation-sensitive defects affecting the compartmentalization and metabolism of Compound A. 1, transporter-mediated movement of A from one compartment to another; 2, defect in the conversion of B to C; 3, increased conversion of B to D caused by accumulation of B; 4, defect in the interaction between an apoenzyme and an obligatory cofactor; 5, decreased feedback inhibition of the conversion of A_in to B as a result of deficiency of C; and 6, secondary inhibition of the conversion of E to F caused by accumulation of D.

Disease results from point defects in metabolism

The signs and symptoms of disease in patients with inborn errors of metabolism are the result of metabolic disturbances caused by deficiency of some catalytic or transport protein. Figure 1.1 shows schematically the relationship between various types of defects and their pathophysiologically and diagnostically important consequences.

Accumulation of substrate

Accumulation of the substrate of a mutant enzyme is an important cause of disease in many inborn errors of metabolism, particularly those involving strictly degradative processes. Some examples are shown in Table 1.1.

Accumulation of substrate is also diagnostically important. Specific diagnosis often follows quickly after identification of the accumulation of metabolites proximal to an enzyme defect, particularly among inborn errors of water-soluble substrates. This is generally true, for example, of the amino acidopathies and organic acidopathies, in which accumulation of substrate throughout the body is often massive, and is reflected by changes in plasma and urine.

In inborn errors of metabolism involving water-insoluble substrates, such as complex lipids, accumulation of the immediate substrate of the mutant enzyme is also important in the pathophysiology of disease. However, accumulation of the compounds is often limited to single tissues or organs, such as brain, which are relatively inaccessible. Moreover, chemical isolation and identification of the metabolites is often cumbersome, requiring laboratory expertise that is not routinely available.

In other disorders, such as the mucopolysaccharide storage diseases, the accumulation of substrate is a major factor in the pathophysiology of disease. However, because the metabolism of the substrate requires the participation of a number of different enzymes, any of which may be deficient as a result of mutation, the demonstration of accumulation is diagnostically important only to the extent that it indicates a class of disorders, not one specific disease. The demonstration of mucopolysaccharide accumulation is important as a screening test for inherited defects in mucopolysaccharide metabolism. However, the metabolism of the individual mucopolysaccharides involves 10 or more genetically distinct lysosomal enzymes, and accumulation of the same compound may occur as a consequence of deficiency of any one of the enzymes. Specific diagnosis in these disorders requires the demonstration of the specific enzyme deficiency in appropriate tissues, such as peripheral blood leukocytes or cultured skin fibroblasts (see Chapter 9).

Accumulation of a normally minor metabolite

In some disorders, the primary cause of disease is accumulation of a normally minor metabolite, produced in excess by a reaction that is usually of trivial metabolic importance. The cataracts in patients with untreated galactosemia occur as a result of accumulation the sugar alcohol, galactitol, a normally minor metabolite of galactose. In another example, accumulation of the normally minor complex lipid metabolite, psychosine, in the brain of infants with Krabbe globoid cell leukodystrophy excites a subacute inflammatory reaction, manifested by appearance in the brain of multinucleated giant cells, called globoid cells. It also causes rapid, severe demyelination, out of proportion to the accumulation of galactocerebroside, the immediate precursor of the defective enzyme, galactocerebrosidase.

Deficiency of product

Deficiency of the product of a specific reaction is another primary consequence of many inherited metabolic diseases. The extent to which it contributes to disease depends on the importance of the product. For example, most of the pathologic consequences of defects of biosynthesis are traceable to deficiency of the product of the relevant reaction - in these cases substrate accumulation plays little or no role in the development of disease. Table 1.2 shows a list of some conditions in which symptoms are the result of deficiency of the product of some enzymic reaction or transport process.

Among the inborn errors of amino acid biosynthesis, the signs of disease are often the combined result of substrate accumulation and product deficiency. For example, in the urea cycle disorder, argininosuccinic aciduria, the defect in the conversion of argininosuccinic acid to arginine causes arginine deficiency, and this, in turn, results in a deficiency of ornithine. Depletion of intramitochondrial ornithine causes accumulation of carbamoylphosphate and ammonia resulting in marked hyperammonemic encephalopathy. The importance of arginine deficiency in the pathophysiology of the encephalopathy is shown by the dramatic response to administration of a single large dose of arginine (4 mmoles/kg given intravenously).

Deficiency of products of reactions is important in two other situations that are common among the inborn errors of metabolism. One of these could be regarded as the result of a 'metabolic steal', a term used to explain the occurrence of myopathy in some patients with glycogen storage disease due to debrancher enzyme deficiency. It was postulated that increased gluconeogenesis in patients with the disease causes accelerated muscle protein breakdown as free amino acids are diverted from protein biosynthesis to gluconeogenesis in an effort to maintain the blood glucose in the face of impaired glycogen breakdown. Another example of the consequences of a metabolic steal is the occurrence of hypoglycemia in patients with hereditary defects in fatty acid oxidation. The over-utilization of glucose and resulting hypoglycemia are a consequence of the inability to meet energy requirements by fatty acid oxidation because of deficiency of one of the enzymes involved in the process.

Another mechanism by which a metabolic defect causes symptoms because of deficiency or inaccessibility of a product might be called 'metabolic sequestration'. Transport defects caused by mutations affecting proteins involved in carrier- mediated transport often produce disease through a failure of the transfer of a meta- bolite from one subcellular compartment to another. The HHH syndrome, named for the associated hyperammonemia, hyperornithinemia, and homocitrullinemia, is caused by a defect in the transport of the amino acid, ornithine, into the

mitochondria. The resulting intramitochondrial ornithine deficiency causes accu- mulation of carbamoylphosphate and ammonia, ultimately causing hyperammonemic encephalopathy, in a manner similar to that causing the hyperammonemia in argininosuccinic aciduria described above.

Secondary metabolic phenomena

Because of the close relationship between the various processes comprising intermediary metabolism, enzyme deficiencies or transport defects inevitably have effects beyond the immediate changes in the concentrations of substrate and product of any particular reaction. These secondary metabolic phenomena often cause diagnostic confusion. For example, ketotic hyperglycinemia was initially thought to be a primary disorder of glycine metabolism. However, subsequent studies showed that glycine accumulation was actually a secondary metabolic phenomenon in patients with a primary defect of propionic acid metabolism. Furthermore, the acute forms of other organic acidopathies, such as methylmalonic acidemia (see Chapter 3), were also found to be associated with marked accumulation of glycine, severe ketoacidosis, and hyperammonemia, all the result of secondary metabolic effects of organic acid or organic acyl-CoA accumulation. Table 1.3 lists some examples of potentially confusing secondary metabolic responses to point defects in metabolism.

Inborn errors of metabolism are inherited

Determination of the pattern of inheritance of a condition is often helpful in making a diagnosis of genetic disease, and it provides the foundation for genetic counselling. The most important information required for establishing the pattern of inheritance is a family history covering at least three generations of relations.

Autosomal recessive disorders

Most of the inherited metabolic diseases recognized today are inherited in the same manner as Garrod's original inborn errors of metabolism: they are Mendelian, single-gene defects, transmitted in an autosomal recessive manner. Disease expression requires that an individual be homozygous for significant, though not necessarily the same, mutations in the same gene. In the overwhelming majority of cases, homozygosity occurs as a result of inheritance of a mutant gene from each parent, who are both heterozygous for the defect. Although it is theoretically possible for one, or even both, of the mutations to arise in the patient as a result of de novo mutation, this is so unlikely that for practical purposes it is ignored. Inheritance of two copies of a mutation from one heterozygous parent may occur as a result of uniparental isodisomy. However, this phenomenon is very rare.