6.1 Clinical Features and Laboratory Investigations
Metachromatic leukodystrophy (MLD) is an autosomal recessive progressive disorder. Its incidence is estimated to be 1:40,000. The disease can be divided into three subtypes: the late infantile (40% of the patients with MLD), juvenile (40%), and adult (20%) variants. This subdivision is based on the age at onset, the duration, and the clinical picture of the disease. As a rule, only one variant of MLD occurs within one family, although exceptions have been reported.
The age of onset in the late infantile variant varies between 6 months and 3 years. Of the different subtypes, this variant is the one that shows the greatest uniformity with regard to clinical picture and course of disease. Most children learn to sit and walk with support normally, but show a delay in walking unaided. The first symptom is usually an unsteady gait due to muscle hypotonia. There are signs of a progressive polyneuropathy, ending in a generalized flaccid paresis of the arms and legs and a loss of tendon reflexes. The neuropathy may be painful. Cerebellar ataxia is also often an early sign. Nystagmus is usually present. Speech development is disturbed and dysarthria becomes manifest. Mental development stagnates and regression occurs. Gradually, involvement of the pyramidal system causes the flaccid paresis to be superseded by a spastic tetraplegia with abnormal reflexes, such as extensor plantar reflexes, but deep tendon reflexes are absent. Bulbar and pseudobulbar symptoms develop, leading to feeding difficulties. Speech is affected, and the child eventually becomes mute. Optic atrophy with impaired vision is present. Epileptic seizures occur in about 25% of the children. Eventually the child becomes blind and completely tetraplegic in a decerebrate state without purposeful movements. This final stage may last for several years. Death usually occurs about 5 years after the onset of clinical symptoms.
The age of onset in the juvenile variant ranges from 4 to 16 years. The disease occurs in apparently healthy,intellectually normal children. Early signs are a gradual deterioration in school performance, language regression, and clumsiness. There are usually also emotional and behavioral disturbances. These symptoms may be present for several months and up to a year before the onset of other neurological signs. A spastic paresis and cerebellar ataxia gradually de velop. On rare occasions the clinical picture is dominated by extrapyramidal features. Clinical symptoms of a peripheral neuropathy are often lacking, and deep tendon reflexes are usually brisk. Optic atrophy develops. Seizures occur in about 50% of the patients. Eventually complete tetraplegia with decerebration posture, brain stem dysfunction, and profound dementia evolves. Death usually occurs 5-10 years after onset.
Some authors prefer to divide the juvenile variant into two subgroups. An early juvenile variant has its onset between 4 and 6 years. The clinical symptomatology resembles that of the late infantile variant, showing gait disturbance and other motor dysfunction as early manifestations. The late juvenile variant has its onset between 6 and 16 years. In the clinical symptomatology, behavioral abnormalities, poor school performance, and language regression predominate as early abnormalities.
The adult form usually reveals itself between 16 and 30 years. Onset of the disease at 60 years or later has also been described. Most patients experience a gradual decline in intellectual abilities. At onset the clinical picture is often dominated by emotional lability, behavioral abnormalities, or psychiatric symptoms such as delusions and hallucinations. It is not uncommon for the patient to be treated initially for schizophrenia or a psychotic depression. After several months or years progressive spastic paresis of the arms and legs develops, with increased tendon reflexes and extensor plantar reflexes. Cerebellar ataxia and such extrapyramidal features as choreiform movements and dystonia may be present. Signs of peripheral neuropathy are often absent, although flaccid tetraparesis may occur in the terminal stage. Optic atrophy and signs of bulbar dysfunction may appear. Epileptic seizures are rare. A state of severe dementia gradually develops. The patient loses contact with the surroundings and lies in a decorticate or decerebrate posture; eventually a persistent vegetative state is reached. The duration of the disease varies from a few years to 15 years and longer. Although a rapid deterioration is seen in some patients, in most cases progression is slow over a period of years. Another, more rare presentation in adults is dominated by signs of a peripheral neuropathy.
CSF protein is elevated in the late infantile form and in most juvenile cases. In the adult form, CSF protein this is less often the case. The EEG is normal at the beginning of the disease. At later stages it shows nonspecific abnormalities in the form of slowing of the background pattern, often together with paroxysmal or epileptiform activity. In the infantile and juvenile variants, the conduction velocity of peripheral nerves is markedly reduced. Particularly in adults, however, the nerve conduction velocity may be normal.
In all patients urinary sulfatide excretion is increased. The activity of arylsulfatase A in urine and in peripheral leukocytes is low,but may be normal in exceptional cases of activator deficiency (see below). In all cases of MLD a decreased catabolism of exogenous sulfatide by cultured fibroblasts can be demonstrated. In this test sulfatide is radiolabeled and added to cultured fibroblasts. The uptake of label by cells with subsequent metabolism is measured at frequent intervals. This procedure is not performed routinely, but only in specific situations. The definitive diagnosis of MLD is based on the determination of the activity of arylsulfatase A in peripheral leukocytes or fi-broblasts. The diagnostic reliability of this test is hampered by the relatively frequent occurrence of so-called pseudo deficiency. In this condition low arylsulfatase A activity is found without associated clinical signs of MLD (see also section 6.4). For this reason, sulfatide excretion in urine should be used to confirm the diagnosis of MLD. It is increased in all cases of MLD, but normal in pseudodeficiency. DNA analysis is another option.
In cases in which symptoms are strongly suggestive of MLD and the enzyme activity in peripheral leukocytes is normal, deficiency of arylsulfatase A activator protein can be surmised. Additional tests then include the measurement of urinary sulfatides and the assessment of fibroblast sulfatide catabolism. A sural nerve biopsy can be performed to demonstrate the deposition of metachromatic material.
Determination of the enzyme activity in cultured chorionic villi or cultured amniotic fluid cells allows prenatal diagnosis. Assessment of sulfatide catabo-lism can be performed in amniotic fluid cells or fetal fibroblasts. DNA-based prenatal diagnosis is also an option. The detection of heterozygotes by determining enzyme activity in leukocytes and DNA techniques facilitates genetic counseling.
Gross inspection of the brain reveals quite a firm consistency in most cases. The brain may be enlarged and heavier than normal, but in later stages reduced size is usually found. The cut surface shows discoloration of the white matter.
Initially, the involvement of the white matter in MLD is patchy, but after some time all the white matter is affected, often in a symmetrical fashion, so that the demyelinated lesions of the two hemispheres have a butterfly configuration. Sometimes, in cases of long duration, the white matter is reduced to a narrow strip 1-2 cm in diameter and shrinkage of the white matter has led to enlargement of the ventricles. De-myelination occurs predominantly in the cerebral hemispheres, especially in the centrum semiovale. Demyelination tends to be most intense in the periventricular area, diminishing towards the surface; the arcuate fibers are relatively spared. The internal capsule, cerebral peduncles in the midbrain, pyramidal tracts in the pons, and pyramids in the medulla are severely affected, but other brain stem tracts are usually only moderately or slightly demyelinated. The cerebellar white matter can also be demyelinated, but usually less so than the cerebral white matter.
Microscopic examination shows demyelination with paucity or complete loss of myelin from affected areas. Axons are relatively spared, but their density is reduced in severely affected areas. There is proliferation of astrocytes with fibrous gliosis. At the edge of affected areas and scattered throughout the lesions there are macrophages that contain the specific degradation products. Usually oligodendroglia are absent from lesions and are reduced in number even in areas where the myelin is still intact. No inflammatory cells are present in the lesions.
MLD is characterized by the deposition of metachromatically staining material in the white matter. The term 'metachromasia' designates the phenomenon of certain cationic dyes changing their color from blue to pink or brown when bound to certain anionic groups present in several organic compounds. In MLD, sulfatides are the organic compounds responsible for the metachromasia. The metachromatic material is mainly stored in the cytoplasm of the proliferated glial cells and macrophages, although some is also found in the oligodendroglia cells,in the neurons of cranial nerve nuclei,basal ganglia, and spinal cord, and seemingly extracellularly as free granules in the white matter. The greatest density of metachromatic deposits is seen in macrophages in perivascular spaces.
The cerebral cortex is relatively intact. Loss of neurons is slight or absent. The cortical neurons contain no metachromatic material. The cerebellar cortex is normal or may show a diffuse loss of granular cells. A decrease in the number of Purkinje cells may occur. In certain areas, metachromatic granular material is stored in the neuronal perikarya, although rarely in large amounts. Neuronal metachromatic deposits are preferentially found in the globus pallidus, thalamus, subthalamic nucleus, hypothalamus, geniculate nucleus, amygdala, and dentate nucleus. The cerebral and cerebellar cortex, claustrum, and caudate nucleus, and also certain brain stem nuclei, tend to be spared.
Electron microscopy demonstrates inclusions bounded by a membrane of lysosomal origin. The morphological organization of the material varies in appearance, probably because of a difference in the lipid composition or in the physicochemical state of the lipids. Prismatic and tuffstone-like profiles are characteristic. In addition to the inclusions that stain metachromatically on light microscopic examination, lamellar inclusions are present in glial cells formed from fragments of degenerated myelin. The 'extracellular' deposits of metachromatic material described in light microscopic studies appear to be cytoplasmic processes containing inclusions in electron microscopy.
Not only are there neuropathological changes in MLD, but also visceral abnormalities. Outside the CNS, metachromatic material is found in the liver, spleen and lymph nodes, gallbladder, pancreas, kidneys, adrenal glands, ovaries, ganglion cells of the retina, and leukocytes of peripheral blood and bone marrow. Storage in these organs is limited to certain cell types. Visceral accumulations are not accompanied by further morphological changes or obvious clinical dysfunction. Although MLD becomes manifest only with neurological abnormalities, it is clear that it is a generalized metabolic disorder.
Biochemical analysis of the white matter shows a greatly increased amount of sulfatide with a concomitant decrease in cerebroside as the major chemical abnormality. Whereas in normal white matter the ratio of cerebroside to sulfatide is approximately 4:1, in MLD it can be below 1. There is not only evidence that the membrane-bound deposits seen in this disease contain sulfatide, but also that myelin, which still appears normal ultrastructurally, has an abnormally high sulfatide content. Other biochemical changes in the white matter are a consequence of loss of myelin with a decrease in cholesterol, phospholipid, and gly-colipids other than sulfatides. There is no increase in cholesterol esters. There are relatively few chemical changes in gray matter.
MLD is a sphingolipidosis caused by deficient activity of the lysosomal enzyme arylsulfatase A (= cere-broside-3-sulfate sulfatase = cerebroside-3-sulfate-3-sulfohydrolase = sulfatide sulfatase). This enzyme catalyzes the hydrolysis of sulfatide, the sulfate ester of cerebroside. Desulfation is the first step in the metabolic degradation of sulfatides. Following this sulfate cleavage, cerebroside is then degraded by cere-broside galactosidase.
The three clinical forms of the disease (late infan-tile,juvenile,and adult onset) can be explained in part by different levels of residual enzyme activity. The arylsulfatase A gene, ARSA, is located on chromosome 22q13.3. Many different mutations have been found in the MLD gene. There is evidence of a geno-type-phenotype correlation in MLD. Some mutations have been exclusively associated with either the infantile-onset or the later onset variants of the disease. Patients with two mutations that cause complete loss of enzyme activity always suffer from the early-onset form of the disease; patients with two mutations that lead to a low residual enzyme activity usually have the adult-onset form of the disease,whereas patients who are compound heterozygous for 'severe' and 'mild' mutations usually have intermediate phenotypes.
Individuals have been found with low (approximately 10-20%) arylsulfatase A activity but no clinical abnormalities. This is called arylsulfatase A pseu-dodeficiency. Individuals who are compound heterozygotes for the pseudodeficiency (PD) mutation and an MLD mutation have 6-10% residual arylsulfatase A activity but do not develop MLD. Thus, arylsul-fatase A activities only slightly higher than those encountered in patients with two mild mutations (2-5%) are sufficient to sustain a normal phenotype.
Pseudodeficiency for arylsulfatase A causes diagnostic problems Pseudodeficiency is related to two common polymorphisms, which are usually found together. These mutations can reduce arylsulfatase A activity to 10% of its control level, but do not lead to clinical symptoms. The allele frequency for pseudo-deficiency is much higher (7-15%) than the MLD allele frequency (0.5 %). Because homozygous pseudo-deficiency is frequent (0.5-2% of the population), it is not uncommon for patients with pseudodeficiency and neurological symptoms of unknown origin to be misdiagnosed as having MLD. There are also serious problems with prenatal diagnosis of MLD in families in which the parents carry an MLD allele and a pseudo-deficiency allele: MLD and pseudodeficiency cannot be distinguished on the basis of enzyme activity determinations using artificial substrates. Measurement of sulfatide excretion in urine and assays measuring the in vivo degradation of sulfatide in cultured fibro-
blasts allow the distinction between MLD and pseudo-deficiency. Both are abnormal in MLD and normal in pseudodeficiency. Demonstration of metachromatic material in a sural nerve biopsy is also diagnostic for MLD. Alternatively, the pseudodeficiency allele can be determined directly by DNA techniques, on the basis of knowledge of the underlying sequence alterations. A problem encountered with this technique, however, is that given the high frequency of the pseudodefi-ciency allele, MLD mutations may also occur within the pseudodeficiency allele, rendering it nonfunctional. The frequency of MLD mutations in the normal and the pseudodeficiency allele is similar, so that 0.5% of the pseudodeficiency alleles harbors an MLD mutation. This finding calls for caution in the diagnosis of pseudodeficiency by DNA tests detecting the mutations of the pseudodeficiency allele. There is evidence that pseudodeficiency mutations may contribute to the phenotype when concurrent with disease causing mutations. Mutations that cause the later onset forms of MLD when they occur in the normal arylsulfatase A gene may lead to a more severe form when they occur in the pseudodeficiency gene.
A small subgroup of MLD patients are not deficient in arylsulfatase A, but in an activator protein that is essential for the enzymatic action of arylsulfa-tase A. The gene for the precursor of this protein, PSAP, is located on chromosome 10q22.1. This gene has been shown to code for a large precursor polypeptide, prosaposin, which is processed to yield four different activator proteins. Sphingolipid activator protein B, also called saposin B, SAP-B, or SAP-1, activates the hydrolysis of sulfatide by arylsulfatase A. In addition, it activates the hydrolysis of GM1-gan-glioside and globotriaosyl ceramide by p-galactosi-dase and a-galactosidase, respectively. The protein interacts with the substrate and solubilizes it for enzymatic hydrolysis. The deficiency of SAP-B causes a disease clinically resembling juvenile or late infantile MLD, but with histochemical and ultrastructural evidence of storage of gangliosides and other glyco-sphingolipids. The diagnosis is established in these cases by revealing metachromatic material in sural nerve biopsy, by finding an increased urinary sulfatide excretion, and by demonstrating deficient turnover of sulfatide in the loading test in cultured fibroblasts, all in the presence of normal arylsulfatase A activity. It can be shown in cultured fibroblasts that the defect in sulfatide catabolism can be corrected by adding activator protein. Deficient turnover of the other glycosphingolipids can also be shown in loading tests in fibroblasts.
Sulfatides are membrane lipids. They are important constituents of cell membranes,including myelin sheaths. Within the cell they are present in the mem branes of organelles. Sulfatide is predominantly present in membranes of myelin-producing cells and in myelin. The amount of this substance normally present in membranes of other organs is much lower. As a result of the block in catabolism,sulfatides accumulate in tissues that normally synthesize them. In the first place, they accumulate in membranes of myelin-producing cells and myelin sheaths, and within the lysosomes of these cells. The membrane build-up is basically normal in MLD. It is the membrane turnover that is abnormal. Sulfatide cannot be degraded and is trapped in the membrane. Simultaneously, the cere-broside content decreases as the conversion of sulfatide to cerebroside is impeded.
A number of pathogenetic mechanisms have been invoked to explain the demyelination in MLD. One of them is the fact that the myelin composition in MLD becomes increasingly abnormal and that therefore myelin possibly becomes increasingly unstable. As soon as the disturbance of the normal physicochemi-cal stability reaches a critical point, demyelination starts. Another explanation proposed is that lysoso-mal storage of sulfatides in oligodendroglia and Schwann cells leads to cellular dysfunction and death, resulting in loss of all myelin sheaths maintained by these cells. Indeed, changes in the subcellular organelles, especially an increase in the numbers of lysosomes of these cells, have been observed before the detection of any morphological abnormalities in the myelin sheaths associated with them. A third mechanism that has been proposed is that sulfogalac-tosylsphingosine, a compound closely related to the cytotoxic compound galactosylsphingosine or psy-chosine in globoid cell leukodystrophy, might accumulate and cause the death of oligodendrocytes and Schwann cells. However, there is no evidence for the enzymatic conversion of sulfatide to sulfogalactosyl-sphingosine, and the concentration of this substance is not elevated in MLD.
The sulfatide accumulation in other organs (kidney, liver, pancreas, adrenal, gallbladder and intestinal tract) does not lead to impairment of functions. Only the gallbladder shows progressive functional impairment attributable to sulfatide accumulation, but gallbladder disease does not contribute to the fatal outcome of MLD. The tolerance of these tissues for sulfatides may be related to the fact that these organs have an excretory function and can discharge the accumulating lipid from the cell into urine, bile, or other fluids. Another important factor is that the sulfatide content of the cellular membranes in these organs is normally much lower than that of the myelin membrane, which has a remarkably high content of galactosphingolipids (cerebroside and sulfatide).
Various forms of therapy have been attempted in an effort to alter the natural course of the disease, but with little success. Diets low in vitamin A or in sulfur (both substances are necessary for the synthesis of sulfatide) have failed to have any favorable effects. After intravenous and intrathecal infusion of arylsul-fatase A the enzyme does not enter the brain, and no clinical benefit has been seen. Increasing numbers of patients have received hematopoietic stem cell transplants in attempts to correct their low cerebral aryl-sulfatase A levels and repair or retard their CNS deterioration. The results appear to depend largely on the stage of disease at transplantation and the rate of disease progression. The results of transplantation in the presymptomatic or early symptomatic stage are better than those of transplantation in the fully symptomatic stage of the disease with decreasing verbal and performance IQs. The chances are better in the late-onset cases, in which disease progression is slower. Stabilization and reversal of MRI abnormalities have been described, as has clinical improvement or arrested progression. However, slow progression despite successful engraftment has also been observed. The chances of stabilization or improvement have to be weighed against the possibility of major complications and death during or after the transplantation. The general experience is that hematopoietic stem cell transplantation has little or no beneficial effect on the polyneuropathy. Gene therapy still has to be tested in the clinical situation.
The CT scan findings in MLD are symmetrical, diffuse decreases in the density of cerebral white matter, with little evidence of cerebral atrophy until the later stages. Hypodensity of the cerebellar white matter has been observed less frequently. No contrast enhancement has been found.
Probably the first abnormalities to be noted on MRI are in the corpus callosum (Fig. 6.1). Subsequently, MRI discloses periventricular white matter abnormalities,with a more or less symmetrical distribution. The white matter lesions are highly confluent. In later onset cases involvement is often predominantly frontal, whereas in early-onset cases occipital predominance can be observed. However, in all variants the cerebral white matter tends to become diffusely affected. The arcuate fibers are relatively spared, but become involved in the later stages. Typically, a pattern of radiating stripes with a signal intensity closer to normal is seen within the abnormal cerebral white matter (Fig. 6.2). On microscopic examination, this radiating pattern is explained by the accumulation of products of myelin breakdown in perivascular macrophages and some sparing of myelin sheaths. This pattern is not evident in all cases (Figs. 6.3, 6.4), and it is lacking especially in far advanced cases with serious white matter atrophy (Fig. 6.5). The corpus callosum is invariably affected, connecting the lesions from both sides. Cerebral white matter atrophy occurs in advanced stages (Fig. 6.5). The posterior limb of the internal capsule becomes involved. Brain stem lesions are observed
Fig. 6.1. MRI in a 5 1/2-year-old boy,who was diagnosed with metachromatic leukodystrophy (MLD) and extensive white matter abnormalities 3 years later.The areas of higher signal in the deep parietal white matter are often seen as a nonspecific finding, but the lesion in the splenium of the corpus callosum
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