3.3. DISORDERS ASSOCIATED WITH DEFECTS IN ENZYMES Robbins and Cotran, Pathologic Basis of Disease blz.

158-163 Lysosomal Storage Diseases Lysosomes are key components of the "intracellular digestive tract." They contain a battery of hydrolytic enzymes, which have two special properties. First, they can function in the acid milieu of the lysosomes. Second, these enzymes constitute a special category of secretory proteins that, in contrast to most others, are destined for secretion not into the extracellular fluids but into an intracellular organelle. This latter characteristic requires special processing within the Golgi apparatus, which is reviewed briefly. Similar to all other secretory proteins, lysosomal enzymes (or acid hydrolases, as they are sometimes called) are synthesized in the endoplasmic reticulum and transported to the Golgi apparatus. Within the Golgi complex, they undergo a variety of posttranslational modifications, of which one is worthy of special note. This modification involves the attachment of terminal mannose-6-phosphate groups to some of the oligosaccharide side chains. The phosphorylated mannose residues may be viewed as an "address label" that is recognized by specific receptors found on the inner surface of the Golgi membrane. Lysosomal enzymes bind to these receptors and are thereby segregated from the numerous other secretory proteins within the Golgi. Subsequently, small transport vesicles containing the receptor-bound enzymes are pinched off from the Golgi and proceed to fuse with the lysosomes. Thus, the enzymes are targeted to their intracellular abode, and the vesicles are shuttled back to the Golgi (Fig. 5-11). As indicated later, genetically determined errors in this remarkable sorting mechanism may give rise to one form of lysosomal storage disease.
Figure 5-11 Synthesis and intracellular transport of lysosomal enzymes.

Figure 5-12 Schematic diagram illustrating the pathogenesis of lysosomal storage diseases. In the example shown, a complex substrate is normally degraded by a series of lysosomal enzymes (A, B, and C) into soluble end products. If there is a deficiency or malfunction of one of the enzymes (e.g., B), catabolism is incomplete and insoluble intermediates accumulate in the lysosomes.

The lysosomal acid hydrolases catalyze the breakdown of a variety of complex macromolecules. These large molecules may be derived from the metabolic turnover of intracellular organelles (autophagy), or they may be acquired from outside the cells by phagocytosis (heterophagy). With an inherited deficiency of a functional lysosomal enzyme, catabolism of its substrate remains incomplete, leading to the accumulation of the partially degraded insoluble metabolite within the lysosomes. Stuffed with incompletely digested macromolecules, these organelles become large and numerous enough to interfere with normal cell functions, giving rise to the so-called lysosomal storage disorders (Fig. 5-12). When this category of diseases was first discovered, it was thought that they resulted exclusively from mutations that led to reduced synthesis of lysosomal enzymes ("missing enzyme syndromes"). In the ensuing years, however, research focusing on the molecular pathology of lysosomal storage diseases has led to the discovery of several other defects. Some of these are as follows: Synthesis of a catalytically inactive protein that cross-reacts immunologically with the normal enzyme. Thus, by immunoassays the enzyme levels appear to be normal. Defects in post-translational processing of the enzyme protein. Included in this category is a failure to attach the mannose-6-phosphate "marker," the absence of which prevents the enzyme from following its correct path to the lysosome. Instead the enzyme is secreted outside the cell. Lack of an enzyme activator or protector protein. Lack of a substrate activator protein. In some instances, proteins that react with the substrate to facilitate its hydrolysis may be missing or defective. Lack of a transport protein required for egress of the digested material from the lysosomes.

It should be evident, therefore, that lysosomal storage disorders can result from the lack of any protein essential for the normal function of lysosomes. Several distinctive and separable conditions are included among the lysosomal storage diseases. In general, the distribution of the stored material, and hence the organs affected, is determined by two interrelated factors: (1) the tissue where most of the material to be degraded is found and (2) the location where most of the degradation normally occurs. For example, brain is rich in gangliosides, and hence defective hydrolysis of gangliosides, as occurs in GM1 and GM2 gangliosidoses, results primarily in storage within neurons and neurologic symptoms. Defects in degradation of mucopolysaccharides affect virtually every organ because mucopolysaccharides are widely distributed in the body. Because cells of the mononuclear phagocyte system are especially rich in lysosomes and are involved in the degradation of a variety of substrates, organs rich in phagocytic cells, such as the spleen and liver, are frequently enlarged in several forms of lysosomal storage disorders. The ever-expanding number of lysosomal storage diseases can be divided into rational categories based on the biochemical nature of the accumulated metabolite, thus creating such subgroups as the glycogenoses, sphingolipidoses (lipidoses), mucopolysaccharidoses (MPS), and mucolipidoses. Only one among the many glycogenoses results from a lysosomal enzyme deficiency, and so this family of storage diseases is considered later. Only the most common disorders among the remaining groups are considered here.

Tay-Sachs Disease (GM2 Gangliosidosis: Hexosaminidase -Subunit Deficiency) GM2 gangliosidoses are a group of three lysosomal storage diseases caused by an inability to catabolize GM2 gangliosides. Degradation of GM2 gangliosides requires three polypeptides encoded by three separate loci (Fig. 5-13). The phenotypic effects of mutations affecting these genes are fairly similar because they result from accumulation of GM2 gangliosides. The underlying enzyme defect, however, is different for each. Tay-Sachs disease, the most common form of GM2 gangliosidosis, results from mutations that affect the -subunit locus on chromosome 15 and cause a severe deficiency of hexosaminidase A. This disease is especially prevalent among Jews, particularly among those of Eastern European (Ashkenazic) origin, in whom a carrier rate of 1 in 30 has been reported.

Figure 5-13 The three-gene system required for hexosaminidase A activity and the diseases that result from defects in each of the genes. The function of the activator protein is to bind the ganglioside substrate and present it to the enzyme. (Modified from Sandhoff K, et al: The GM2 gangliosidoses. In Scriver CR, et al [eds]: The Metabolic Basis of Inherited Disease, 6th ed. New York, McGraw-Hill, 1989, p. 1824.)

Morphology. The hexosaminidase A is absent from virtually all the tissues that have been examined, including leukocytes and plasma, and so GM2 ganglioside accumulates in many tissues (e.g., heart, liver, spleen), but the involvement of neurons in the central and autonomic nervous systems and retina dominates the clinical picture. On histologic examination, the neurons are ballooned with cytoplasmic vacuoles, each of which constitutes a markedly distended lysosome filled with gangliosides (Fig. 5-14A). Stains for fat such as oil red O and Sudan black B are positive. With the electron microscope, several types of cytoplasmic inclusions can be visualized, the most prominent being whorled configurations within lysosomes composed of onion-skin layers of membranes (Fig. 5-14B). In time, there is progressive destruction of neurons, proliferation of microglia, and accumulation of complex lipids in phagocytes within the brain substance. A similar process occurs in the cerebellum as well as in neurons throughout the basal ganglia, brain stem, spinal cord, and dorsal root ganglia and in the neurons of the autonomic

nervous system. The ganglion cells in the retina are similarly swollen with GM2 ganglioside, particularly at the margins of the macula. A cherry-red spot thus appears in the macula, representing accentuation of the normal color of the macular choroid contrasted with the pallor produced by the swollen ganglion cells in the remainder of the retina. This finding is characteristic of Tay-Sachs disease and other storage disorders affecting the neurons.

Figure 5-14 Ganglion cells in Tay-Sachs disease. A, Under the light microscope, a large neuron has obvious lipid vacuolation. (Courtesy of Dr. Arthur Weinberg, Department of Pathology, University of Texas Southwestern Medical Center, Dallas.) B, A portion of a neuron under the electron microscope shows prominent lysosomes with whorled configurations. Part of the nucleus is shown above. (Electron micrograph courtesy of Dr. Joe Rutledge, University of Texas Southwestern Medical Center, Dallas, TX.)

Many alleles have been identified at the -subunit locus, each associated with a variable degree of enzyme deficiency and hence with variable clinical manifestations. The affected infants appear normal at birth but begin to manifest signs and symptoms at about age 6 months. There is relentless motor and mental deterioration, beginning with motor incoordination, mental obtundation leading to muscular flaccidity, blindness, and increasing dementia. Sometime during the early course of the disease, the characteristic, but not pathognomonic, cherry-red spot appears in the macula of the eye grounds in almost all patients. Over the span of 1 or 2 years, a complete, pathetic vegetative state is reached, followed by death at age 2 to 3 years. Antenatal diagnosis and carrier detection are possible by enzyme assays and DNA-based analysis.25 The clinical features of the two other forms of GM2 gangliosidosis (see (Fig. 5-13), Sandhoff disease, resulting from -subunit defect, and GM2 activator deficiency, are similar to those of Tay-Sachs disease.

Niemann-Pick Disease: Types A and B. Niemann-Pick disease types A and B refers to two related disorders that are characterized by lysosomal accumulation of sphingomyelin resulting from an inherited deficiency of sphingomyelinase. In the past, these two conditions were grouped with an unrelated disorder, called Niemann-Pick disease type C, described separately below. Type A is the severe infantile form with extensive neurologic involvement, marked visceral accumulations of sphingomyelin, and progressive wasting and early death within the first 3 years of life. To provide a perspective on the differences between the variants of Niemann-Pick disease, we need only point out that in type B, for example, patients have organomegaly but generally no central nervous system involvement. They usually survive into adulthood. As with Tay-Sachs disease, Niemann-Pick disease types A and B are common in Ashkenazi Jews. Morphology. In the classic infantile type A variant, a missense mutation causes almost complete deficiency of sphingomyelinase. Sphingomyelin is a ubiquitous component of cellular (including organellar) membranes, and so the enzyme deficiency blocks degradation of the lipid, resulting in its progressive accumulation within lysosomes, particularly within cells of the mononuclear phagocyte system. Affected cells become enlarged, sometimes to 90 m in diameter, secondary to the distention of lysosomes with sphingomyelin and cholesterol. Innumerable small vacuoles of relatively uniform size are created, imparting a foaminess to the cytoplasm (Fig. 5.15). In frozen sections of fresh tissue, the vacuoles stain for fat with Sudan black B and oil red O. Electron microscopy confirms that the vacuoles are engorged secondary lysosomes that often contain membranous cytoplasmic bodies resembling concentric lamellated myelin figures. Sometimes the lysosomal configurations take the form of parallel palisaded lamellae, creating so-called zebra bodies. The lipid-laden phagocytic foam cells are widely distributed in the spleen, liver, lymph nodes, bone marrow, tonsils, gastrointestinal tract, and lungs. The involvement of the spleen generally produces massive enlargement, sometimes to 10 times its normal weight, but the hepatomegaly is usually not quite so striking. The lymph nodes are generally moderately to markedly enlarged throughout the body. Involvement of the brain and eye deserves special mention. In the brain, the gyri are shrunken and the sulci widened. The neuronal involvement is diffuse, affecting all parts of the nervous system. Vacuolation and ballooning of neurons constitute the dominant histologic change, which in time leads to cell death and loss of brain substance. A retinal cherry-red spot similar to that seen in Tay-Sachs disease is present in about one third to one half of affected individuals. Its origin is similar to that described in Tay-Sachs disease except that the accumulated metabolite is sphingomyelin.

Figure 5-15 Niemann-Pick disease in liver. The hepatocytes and Kupffer cells have a foamy, vacuolated appearance owing to deposition of lipids. (Courtesy of Dr. Arthur Weinberg, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX.)

Clinical manifestations may be present at birth but almost invariably become evident by age 6 months. Infants typically have a protuberant abdomen because of the hepatosplenomegaly. Once the manifestations appear, they are followed by progressive failure to thrive, vomiting, fever, and generalized lymphadenopathy as well as progressive deterioration of psychomotor function. Death comes as a release, usually within the first or second year of life. The diagnosis is established by biochemical assays for sphingomyelinase activity in liver or bone marrow biopsy. The sphingomyelinase gene has been cloned, and hence individuals affected with types A and B as well as carriers can be detected by DNA analysis. Nieman-Pick Disease: Type C (NPC). Although discovered more recently as a distinct entity, this type is more common than types A and B combined. The affected gene, called NPC-1, encodes a protein involved in cholesterol trafficking; therefore, in this disorder, cholesterol accumulates in the affected cells. In the brain, NPC-1 is present in astrocytic processes in close apposition to nerve terminals. With defective cholesterol trafficking, terminal axons and dendrites degenerate. NPC is clinically very heterogeneous. It may present with hydrops fetalis and stillbirth, as neonatal hepatitis, or as a chronic form characterized by progressive neurologic damage. Most common, however, is presentation in childhood, which is marked by ataxia, supranuclear palsy, psychomotor regression, hepatosplenomegaly, and dysarthria.