Child Neurology
7th Edition

Phenylketonuria is an autosomal recessive disorder that results from a mutation in the gene that codes for phenylalanine hydroxylase (PAH), the enzyme that hydroxylates phenylalanine to tyrosine. The complete hydroxylation system consists in part of PAH, a PAH stabilizer, the tetrahydrobiopterin cofactor (BH4), dihydropteridine reductase (DHPR), which is required to recycle BH4, and a BH4-synthesizing system. This system involves guanosine triphosphate (GTP) cyclohydrolase and 6-pyruvoyl tetrahydropteridine synthetase (Fig. 1.1) (14).

PAH is normally found in liver, kidney, and pancreas but not in brain or skin fibroblasts. The enzyme is an iron-containing metalloprotein dimer of identical subunits with a molecular weight of approximately 100,000. The gene coding for the enzyme has been cloned and localized to the long arm of chromosome 12 (12q22–q24). The gene is approximately 90 kilobases (kb) long with 13 exons, and codes for a mature RNA of approximately 2,400 bases. Availability of this clone has facilitated the molecular genetic analysis of patients with PKU and has confirmed that PKU is the consequence of numerous mutant alleles arising in various ethnic groups. The majority of these mutations result in deficient enzyme activity and cause hyperphenylalaninemia. Mutations occur in all 13 exons of the gene and flanking sequence. Some cause phenylketonuria; others cause non-PKU hyperphenylalaninemia, whereas still others are silent polymorphisms present on both
normal and mutant chromosomes (15,16). Some PAH alleles are more prevalent; five account for approximately 60% of European mutations and they tend to cluster in regions or are on only one of a few haplotypes. More than 495 mutations have been recorded. They are catalogued at http://www.pahdb.mcgill.ca.

Most mutations are missense mutations, although splice, nonsense, and silent mutations as well as single–base-pair frameshifts and larger deletions and insertions have been found. Most patients with PKU are compound heterozygotes rather than homozygotes in the precise meaning of the term (17). As a rule, the biochemical phenotype (i.e., the degree of phenylalanine elevation) does not correlate with the activity of PAH as predicted from the genetic mutation. In addition, no good correlation exists between PAH activity and intellectual function of untreated patients (18). Scriver (3,5) and others have pointed out that various environmental factors and modifying genes, such as the blood–brain barrier phenylalanine transporters, and variations in brain phenylalanine consumption rate may affect intellectual function in phenylketonuric patients (19,20).

The infant with classic PKU is born with only slightly elevated phenylalanine blood levels, but because of the absence of PAH activity, the amino acid derived from food proteins and postnatal catabolism accumulates in serum, CSF, and brain and is excreted in large quantities. In lieu of the normal degradative pathway, phenylalanine is converted to phenylpyruvic acid, phenylacetic acid, and phenylacetylglutamine.

The transamination of phenylalanine to phenylpyruvic acid is sometimes deficient for the first few days of life, and the age at which phenylpyruvic acid can be first detected varies from 2 to 34 days. From the first week of life, o-hydroxyphenylacetic acid also is excreted in large amounts.

In addition to the disruption of phenylalanine metabolism, tryptophan and tyrosine are handled abnormally. Normally, transport across the blood–brain barrier of a large neutral amino acid such as phenylalanine is determined by its plasma concentration and its affinity to the stereo-specific L-type amino acid carrier system (20). Supraphysiologic levels of phenylalanine competitively inhibit transport of tryptophan and tyrosine across the blood–brain barrier (21). Intestinal transport of L-tryptophan and tyrosine is also impaired in PKU, and fecal content of tryptophan and tyrosine is increased. These abnormalities are reversed by dietary correction of the plasma phenylalanine levels (22). Miyamoto and Fitzpatrick suggested that a similar interference might occur in the oxidation of tyrosine to 3-(3,4-dihydroxyphenyl) alanine (dopa), a melanin precursor, and might be responsible for the deficiency of hair and skin pigment in phenylketonuric individuals (23). The biochemical pathology of PKU is reviewed to a greater extent by Scriver and Kaufman (14).

Alterations within the brain are nonspecific and diffuse. They involve gray and white matter, and they probably progress with age. Three types of alterations exist.

Phenylketonuric infants appear healthy at birth. In untreated infants, vomiting, which at times is projectile, and irritability are frequent during the first 2 months of life. By 4 to 9 months, delayed intellectual development becomes apparent (28). In the untreated classic case, mental retardation is severe, precluding speech and toilet training. Children in this category have an IQ below 50. Seizures, common in the more severely retarded, usually start before 18 months of age and can cease spontaneously. During
infancy, they often take the form of infantile spasms, later changing into tonic-clonic attacks.

The untreated phenylketonuric child is blond and blue-eyed, with normal and often pleasant features. The skin is rough and dry, sometimes with eczema. A peculiar musty odor, attributable to phenylacetic acid, can suggest the diagnosis. Significant neurologic abnormalities are rare, although hyperactivity and autistic features are not unusual. Microcephaly may be present as well as a mild increase in muscle tone, particularly in the lower extremities. A fine, irregular tremor of the outstretched hands is seen in approximately 30% of the patients. Parkinsonian-like extrapyramidal symptoms also have been encountered (29). The plantar response is often extensor.

A variety of electroencephalographic (EEG) abnormalities has been found, but hypsarrhythmic patterns, recorded even in the absence of seizures, and single and multiple foci of spike and polyspike discharges are the most common (30).

MRI is abnormal in almost every patient, regardless of when treatment was initiated. On T2-weighted imaging, one sees increased signal in the periventricular and subcortical white matter of the posterior hemispheres. Increased signal can extend to involve the deep white matter of the posterior hemispheres and the anterior hemispheres. No signal abnormalities are seen in brainstem, cerebellum, or cortex, although cortical atrophy may be present (Fig. 1.4) (31,32). The severity of the abnormality is unrelated to the patient’s IQ but is significantly associated with the phenylalanine level at the time of imaging. In adult patients with PKU who had come off their diets, resumption of dietary treatment can improve MRI abnormalities within
a few weeks or months, an observation that strongly suggests that at least some of the MRI changes are the result of edema (32).

Heterozygous mothers tend to have elevated plasma phenylalanine levels and somewhat reduced tyrosine values during the latter part of pregnancy and after delivery. Mothers suffering from PKU have a high incidence of spontaneous abortions. It is now clear that when maternal blood phenylalanine levels are greater than 20 mg/dL (1,212 μmol/L) during pregnancy, fetal brain damage is almost inevitable, with mental retardation encountered in 92% of offspring and microcephaly in 73%. Offspring have an unusual facies: upturned nose, underdeveloped philtrum, and a thin upper lip (33). Additionally, a significant incidence of congenital heart disease and prenatal and postnatal growth retardation occurs (34). MRI in these children shows hypoplasia or partial agenesis of the corpus callosum and delayed myelination (35). These observations are best explained by a deleterious effect of elevated phenylalanine on the myelinating ability of oligodendrocytes.

Much, if not all, of the fetal damage appears to be preventable by placing phenylketonuric mothers on a phenylalanine-restricted diet before conception and maintaining blood phenylalanine levels below 6 mg/dL (360 μmol/L) throughout pregnancy (36). In the data compiled by Koch and coworkers the optimal outcome, as measured by the Wechsler Intelligence Scale for Children (WISC) score of offspring obtained at 7 years of age, was obtained when maternal blood phenylalanine levels of 120 to 360 μmol/L (2 to 6 mg/dL) were obtained by 8 to 10 weeks’ gestation and maintained at those levels throughout pregnancy. However, there was considerable scatter in IQ even when the phenylalanine levels were maintained below 5 mg/dL (37). Birth weight and head circumference in infants of mothers so managed were normal, and no evidence existed for fetal nutritional deficiency. Offspring of mothers who experience mild PKU as defined by phenylalanine levels of less than 400 μmol/L (6.6 mg/dL) appear to be normal, and mothers do not require dietary intervention (38).

The pathogenesis of mental retardation in PKU is not completely understood (39). No evidence exists that phenylpyruvic acid or any of the other phenylalanine metabolites are neurotoxic at concentrations in which they are seen in PKU (40). Probably no single factor is responsible; instead, impairment of amino acid transport across the blood–brain barrier; disruption of the brain amino acid pool; with consequent defective proteolipid protein synthesis, impaired myelination, and low levels of neurotransmitters, such as serotonin, are responsible to varying degrees (41,42).

With the worldwide practice of neonatal screening for phenylketonuria, the diagnosis is usually made during the first week of life, and it is now rare to encounter an older infant or child with undiagnosed PKU. As mentioned in the section on Molecular Genetics and Biochemical Pathology, plasma phenylalanine levels are elevated in the cord blood of phenylketonuric infants and increase rapidly within a few hours of birth. A screening program that involves spectrofluorometric or microbiologic estimation of blood phenylalanine is used commonly. Tandem mass spectrometric analysis of phenylalanine and other amino acids as well as of the acylcarnitines of the various organic acids is being used in some laboratories (43), and trials of this method for newborn screening are being instituted in several states. Whatever the method, the screening test requires a drop of whole blood placed on a filter paper. If the test is positive [a value greater than 2 to 4 mg/dL (120 to 240 μmol/L)], the diagnosis is confirmed by quantitative analysis of phenylalanine and tyrosine in blood. Although most infants whose blood phenylalanine levels are above the threshold value do not have PKU, such patients require prompt reevaluation by an appropriate laboratory to determine whether hyperphenylalaninemia is persistent and whether it is caused by PAH deficiency.

Of considerably greater concern are the false-negative test results. It is now clear that routine screening tests, when correctly performed after 12 hours of life, detect all infants with classic PKU. In a group of such infants studied by Koch and Friedman the lowest phenylalanine value recorded at 24 hours was 5.6 mg/dL (339 μmol/L); at 48 hours, 7.5 mg/dL (454 μmol/L); and at 72 hours, 8.4 mg/dL (509 μmol/L). Thus, even at 24 hours, none of the infants with classic PKU would have escaped detection (44). The Canadian experience of Hanley and coworkers is similar (45). As a rule, breast-fed PKU infants have higher phenylalanine levels than those who are formula fed. In some infants with mild hyperphenylalaninemia, the increase in blood phenylalanine is sufficiently slow to allow them to escape detection unless an assay is used, such as tandem mass spectroscopy, that can measure both phenylalanine and tyrosine, which decreases false-positive results. Some screening agencies obtain a second screening blood specimen from all infants whose first blood specimen was drawn during the first 24 hours of life (46,47).

Most infants with elevated blood phenylalanine detected by means of the newborn screening program do not have classic PKU; instead they have a transient or mild form of hyperphenylalaninemia that is usually benign. Patients with mild PKU have phenylalanine levels between 600 and 1200 μmol/L (10 to 20 mg/dL) on an unrestricted diet, and patients with mild hyperphenylalaninemia have levels below 600 μmol/L (10 mg/dl) on an unrestricted diet (48). A large proportion of these patients represent compound heterozygotes for a mutation that abolishes catalytic activity of PAH and one that reduces it (16,49,50,51). With normal protein intakes, the majority of such infants appear to have unimpaired intelligence, even when untreated (48).

Elevated blood phenylalanine levels also are observed in 25% of premature infants and occasionally in full-term newborns. In all of these patients, tyrosine levels are increased to a much greater extent than are phenylalanine levels.

Prenatal diagnosis can be performed by detection of the specific mutations in PAH identified in the family or by genetic linkage analysis (52,53). Modifier genes have not been identified.

Two treatment strategies could be employed in PKU: modification of the phenotypic expression of the defective gene and definitive treatment by correcting the gene defect. Only the first approach has been used in clinical practice.

On referral of an infant with a confirmed positive screening test result, the first step is quantitative determination of serum phenylalanine and tyrosine levels. All infants whose blood phenylalanine concentration is greater than 10 mg/dL (600 μmol/L) and whose tyrosine concentration is low or normal (1 to 4 mg/dL) should be started on a low-phenylalanine diet immediately. Infants whose blood phenylalanine concentrations remain below 10 mg/dL (600 μmol/L) on an unrestricted diet are generally considered not to require a diet (54).

The accepted therapy for classic PKU is restriction of the dietary intake of phenylalanine by placing the infant on one of several low-phenylalanine formulas. The diet should be managed by a team consisting of a nutritionist, a physician with expertise in metabolic disorders, and a person to ensure dietary compliance. To avoid symptoms of phenylalanine deficiency, regular formula is added to the diet in amounts sufficient to maintain blood levels of the amino acid between 2 and 6 mg/dL (120 and 360 μmol/L). Generally, patients tolerate this diet quite well, and within 1 to 2 weeks, the serum concentration of phenylalanine becomes normal. Serum phenylalanine determinations are essential to ensure adequate regulation of diet. These are performed twice weekly during the first 6 months of life and twice monthly thereafter.

Strict dietary control should be maintained for as long as possible, and most centers strive to keep levels below 6.0 mg/dL (360 μmol/L), even in patients with moderate and mild PKU. Samples of low-phenylalanine menus are given by Buist and colleagues (55), and the nutritional problems inherent in prolonged use of a restricted diet are discussed by Acosta and colleagues (56). Dietary lapses are common, particularly in patients 15 years or older (57). They frequently are accompanied by progressive white
matter abnormalities on MRI. Some workers have suggested supplementation of the low-phenylalanine diet with tyrosine, but no statistical evidence exists that this regimen results in a better intellectual outcome. This possibly could be due to failure to achieve adequate tyrosine levels in the brain even with tyrosine supplementation of 300 mg/kg (58). Failure to treat patients with mild hyperphenylalaninemias does not appear to produce either intellectual deficits or MRI abnormalities.

Patients with mild PKU or mild hyperphenylalaninemia have been treated successfully with tetrahydrobiopterin (BH4; 10 to 20 mg/kg per day) (59,59a). Most of these individuals have at least one missense mutation in the gene for phenylalanine hydroxylase, leading to some residual enzyme activity (60). The mechanism for this effect is unknown, as is the question of how to best select patients responsive to the cofactor. BH4 is being tested, but it is not yet approved for therapy in the United States.

Dietary therapy has for the greater part been effective in preventing mental retardation in patients with severe PKU. It has become apparent that the outcome depends on several factors. Most important is the age at which the diet was initiated. Smith and coworkers found that patients’ IQ fell approximately 4 points for each month between birth and start of treatment (61). The average phenylalanine concentration while receiving treatment also affects outcome, with optimal average phenylalanine levels in the most recent cohorts being 5.0 to 6.6 mg/dL (300 to 400 μmol/L). Additionally, hypophenylalaninemia during the first 2 years of life [i.e., the length of time that phenylalanine concentration was below 2.0 mg/dL (120 μmol/L)] also affected outcome adversely. Even patients with normal IQ scores and with the most favorable diagnostic and treatment characteristics have lower IQs than other members of their families and suffer from cognitive deficits, educational difficulties, and behavioral problems, notably hyperactivity (61,62,63). As was already noted, dietary supplementation with tyrosine, although used in several centers, does not appear to improve performance on neuropsychologic tests (64). The most likely explanations for these deficits are inadequate dietary control and unavoidable prenatal brain damage from elevated phenylalanine (see Fig. 1.2) (24).

When patients who already have developed symptoms caused by classic PKU are placed and maintained on a low-phenylalanine diet, the epilepsy comes under control and their EEGs normalize. Microcephaly, if present, can correct itself, and abnormally blond hair regains its natural color.

Considerable uncertainty exists about when, if ever, to terminate the diet (65). In the series of Waisbren and coworkers (66), one-third of youngsters whose diet was discontinued at 5 years of age had a reduction in IQ of 10 points or more during the ensuing 5 years. The blood phenylalanine level when the children were off the restrictive diet predicted the change in IQ. Of the children whose IQs dropped 20 or more points, 90% had blood phenylalanine levels of 18 mg/dL (1,090 μmol/L) or higher, and 40% of those whose IQs rose 10 points or more had a phenylalanine level of less than 18 mg/dL (1,090 μmol/L). In young adult patients with PKU, discontinuation of the diet was accompanied by progressive spasticity and worsening white matter abnormalities (67). Reinstitution of the diet results in clear clinical improvement and resolution of new MRI abnormalities. Reports such as these speak against early relaxation of the restrictions on phenylalanine intake and indicate that dietary therapy for patients with classical PKU should be life-long (68,69).

The relative inadequacies of dietary therapy underscore the need for a more definitive approach to the treatment of PKU. Allogeneic or autologous bone marrow transplants are being used for the treatment of a variety of genetic diseases (Table 1.7). The likelihood that this procedure will cure or at least stabilize a genetic disease depends on the tissue-specific expression of the normal gene product, the patient’s clinical symptoms, and the cellular transport of the normal gene product. In PKU, the defective enzyme is not normally expressed in bone marrow–derived cells, and bone marrow transplantation has no therapeutic value (see Table 1.7).

Another approach to treating the patient with PKU is the introduction of PAH gene into affected hepatic cells. Recombinant viruses containing human PAH have been introduced into mouse hepatoma cells, where they are able to continue expressing the human enzyme, lowering the phenylalanine level and normalizing coat color (70). The next step would be to find a virus that can infect human liver, maintain itself there without inducing damage to the organ, and allow the human gene to continue functioning in the new host. Ding and colleagues reviewed various approaches for nonviral gene transfer (71).

MSUD is characterized by the accumulation of three branched-chain ketoacids: α-ketoisocaproic acid, α-ketoisovaleric acid, and α-keto-β-methylvaleric acid, the derivatives of leucine, valine, and isoleucine, respectively (104,105). Their accumulation is the consequence of a defect in oxidative decarboxylation of branched-chain ketoacids (Fig. 1.5).

The branched-chain α-ketoacid dehydrogenase complex is located within the mitochondrial inner membrane matrix compartment. It is a multienzyme complex comprising six proteins: E_1α and E_1β. which form the decarboxylase; E₂; E₃; and a branched-chain–specific kinase and phosphatase. The last two enzymes regulate the activity of the complex by phosphorylating and dephosphorylating the dehydrogenase. E₁ is a thiamin pyrophosphate-dependent enzyme. The second enzyme (E₂), dihydrolipoyltransacylase, transfers the acyl group from the first enzyme to coenzyme A. The third enzyme (E₃), dihydrolipoyldehydrogenase, a flavoprotein, reoxidizes the disulfhydryl form of lipoamide. The same enzyme is common to other α-ketoacid dehydrogenases, such as pyruvate and α-ketoglutarate dehydrogenase (106). The complex removes carboxyl groups from all three branched-chain ketoacids and converts those ketoacids to their respective coenzyme A derivatives (see Fig. 1.5, step 2). Chuang and coworkers reviewed the crystal structure of the various enzyme components and the structural basis for the various MSUD mutations (103).

With six genes involved in the function of the branched-chain ketoacid dehydrogenase complex, considerable room for heterogeneity exists. Mutations in the genes for E_1α, E_1β, E₂, and E₃ have been described, with many MSUD patients being compound heterozygotes. These induce a continuum of disease severity that ranges from the severe, classic form of MSUD to mild and intermittent forms.

As a consequence of the enzymatic defect, the branched-chain ketoacids accumulate in serum and CSF and are excreted in large quantities in urine (105). Plasma levels of the respective amino acids (e.g., leucine, isoleucine, and valine) are elevated secondary to the increase in ketoacid concentrations. Alloisoleucine, which is formed by transamination of α-keto-β-methylvaleric acid, also has been found in serum (107). In some cases, the branched-chain hydroxyacids, most prominently α-hydroxyisovaleric acid (108), are excreted also. Sotolone, a derivative of α–ketobutyric acid, the decarboxylation of which is impaired by accumulation of α-keto-β-methylvaleric acid, is responsible for the characteristic odor of the patient’s urine and perspiration (109).

Structural alterations in the nervous system in untreated infants with MSUD are similar to, but more severe than, those seen in PKU. In infants dying during the acute phase of the disease, diffuse edema occurs (101). The cytoarchitecture of the cortex is generally immature, with fewer cortical layers and the persistence of ectopic foci of neuroblasts, an indication of disturbed neuronal migration. Dendritic development is abnormal, and the number of oligodendrocytes and the amount of myelin are less than would be seen in a healthy brain of comparable age (110). Marked astrocytic gliosis and generalized cystic degeneration occur (111). Little clinical or pathologic evidence exists for demyelination in patients who are treated early (112,113). On chemical examination, the concentration of myelin lipids is markedly reduced, with cerebrosides, sulfatides, and proteolipid protein almost completely absent. These abnormalities are not found in infants dying of the disease within the first days of life or in patients treated by restriction of branched-chain amino acid intake (113).

Various mutations result in five fairly distinct clinical phenotypes of MSUD. Patients can be homozygotes for the same allele or compound heterozygotes for different alleles. The classic form of MSUD accounts for approximately 75% of patients (103). Mutations in the E₁ decarboxylase component of the enzyme are associated with this phenotype (114). In the original four patients reported by Menkes and coworkers (101) as well as in subsequent cases, dystonia, opisthotonos, intermittent increase in muscle tone, and respiratory irregularities appeared within the first week of life in babies apparently healthy at birth (115). Subsequently, rapid deterioration of the nervous system occurred, and all but one died within 1 month. In some patients, cerebral edema is marked and can be fatal (116). Other patients, spastic and intellectually retarded, survived without treatment for several years. A fluctuating ophthalmoplegia correlates in intensity with serum leucine levels (117). Presentation with pseudotumor cerebri also has
been reported. Approximately 50% of patients with the classic form of MSUD develop severe hypoglycemia; this is probably the consequence of a defective gluconeogenesis from amino acids, particularly alanine (118).

MRI during the acute stage of the disease before treatment is characteristic. It demonstrates edema that is maximal in cerebellar deep white matter, the posterior portion of the brainstem, and the posterior limb of the internal capsule. Edema also is seen in the cortical U fibers, the head of the caudate, and the putamen (119,120). These findings are consistent with the location of status spongiosus noted on pathologic examination. The cause of the
acute cerebral edema and its unique localization are unknown. Diffusion MRI suggests that in the myelinated areas of the brain there is an intramyelinic cytotoxic edema, whereas in the unmyelinated areas there is vasogenic interstitial edema (121,122). Because neurologic symptoms become apparent with relatively mild increases in plasma leucine concentrations, whereas there is little apparent toxicity associated with increased levels of isoleucine or valine, damage probably results from leucine accumulation or from accumulation of its ketoacid metabolite. As is the case in PKU, chronic brain damage is probably caused by interference with amino acid transport into the brain, a deranged amino acid environment, and, consequently, failure in biosynthesis of proteolipids, myelin, and neurotransmitters (123). In treated patients, the MRI discloses symmetric bilateral periventricular high signal intensity on T2-weighted images. This picture is similar to that seen in PKU and suggests focal dysmyelination (112).

The intermittent form of MSUD results from a variety of mutations in the gene for E₂, and branched-chain dehydrogenase activity is higher than in the classic form, usually 5% to 20% of normal (124). The clinical picture is that of intermittent periods of ataxia, drowsiness, behavior disturbances, and seizures that make their first appearance between ages 6 and 9 months. Attacks are generally triggered by infections, immunizations, or other forms of stress (125).

In the intermediate form, the clinical picture is one of mild to moderate mental retardation (126). Branched-chain dehydrogenase activity ranges from 5% to 20% of normal, and the defect is usually in the gene coding for E₁ (106).

A thiamin-responsive variant represents an entity in which, in some cases at least, a mutant exists in the gene for E₂ (103). Chuang and coworkers proposed that binding of the mutated, inactive E₂ component to the wild-type E₁ component enhances wild-type E₁ activity, and that the augmented E₁ activity is responsible for the response to thiamine (114).

Mutants defective in the gene for E₃ present with hypotonia, rapid neurologic deterioration, and severe lactic acidosis. This entity is discussed with the various other organic acidemias.

The most common presentation of MSUD is that of a term infant who initially seems to be well for a few days and then deteriorates. The rate of deterioration varies, and most infants initially are believed to be septic. The neurologist called in to consult on such an infant should always consider the diagnosis of an inborn error of metabolism. Basic investigations at this point include blood or plasma pH, blood gases, glucose, electrolytes, liver function tests, ammonia, and plasma for amino acids and acyl carnitines. Urine for sugars, ketones, and organic acids is also indicated (127). In addition to MSUD, neurologic deterioration during the neonatal period is seen in various organic acidurias, urea cycle defects, fatty acid oxidation defects, and the congenital lactic acidoses. Clinically, MSUD is distinguished by the characteristic odor of the patient, a positive urine 2,4-dinitrophenylhydrazine test, and an elevation of the plasma branched-chain amino acids. The characteristic increase in plasma leucine, isoleucine, and valine is seen by the time the infant is 24 hours old, even in those infants who have not yet been given protein (128). Routine newborn screening for the condition using a bacterial inhibition assay analogous to that used for the neonatal diagnosis of PKU is performed in many states in the United States and in many other countries (129). Tandem mass spectroscopy also can be used and has the advantage of obtaining rapid quantitative measurements of all three branched-chain amino acids (43). The presence of the branched-chain ketoacid decarboxylases in cultivated amniocytes and chorionic villi allows the antenatal diagnosis of the disease as early as 10 weeks’ gestation (130).

Treatment consists in inhibiting endogenous protein catabolism, sustaining protein synthesis, preventing deficiencies of essential amino acids, and maintaining normal serum osmolarity (131). Morton and coworkers stressed that restriction of the dietary intake of the branched-chain amino acids through the use of one of several commercially available formulas is secondary in importance to inhibition of protein catabolism and enhancement of protein synthesis (131). For optimal results, infants should be placed on the diet during the first few days of life and should receive frequent measurements of serum amino acids. Prompt and vigorous treatment of even mild infections is mandatory; a number of children on this synthetic diet have died of septicemia (115,131).

Peritoneal dialysis or hemodialysis has been used to correct coma or other acute neurologic symptoms in the newly diagnosed infant (132). Another, simpler approach is to provide intravenously or by nasogastric tube an amino acid mixture devoid of leucine but containing large amounts of tyrosine, glutamine, and alanine (133). Morton and coworkers believe that the brain edema that is frequently seen in the acutely ill neonate results from hyponatremia and responds promptly to addition of oral and intravenous saline (131). Most of the children in whom long-term dietary therapy was initiated during the first 2 weeks of life and whose dietary control was meticulously maintained achieved normal or nearly normal IQs (131,134). In the experience of Hilliges and coworkers, the mean IQ of MSUD patients at 3 to 16 years of age was 74 ± 14, as compared with 101 ± 12 for early-treated patients with PKU. The length of time after birth that plasma leucine
concentrations remain elevated appears to affect the IQ, as does the amount, if any, of residual branched-chain ketoacid dehydrogenase activity (135). The thiamin-responsive child is treated with 10 to 1,000 mg of thiamin per day (103).

Argininosuccinic acid is a normal intermediary metabolite in the synthesis of urea (see Fig. 1.6). A deficiency in argininosuccinate lyase, an enzyme whose gene has been mapped to chromosome 7cn–q11.2, has been demonstrated in liver and skin fibroblast cultures (157).

The synthesis of urea is only slightly depressed, but a large proportion of labeled ammonium lactate administered to affected individuals is converted to glutamine (158). The manner in which children synthesize urea is not clear. It appears likely that in argininosuccinic aciduria, as well as in the other defects of urea cycle, substrate accumulates to a concentration at which the decreased substrate-binding capacity of the mutant enzyme is overcome by the accumulation of precursor to levels greater than the K_M for the mutated enzyme (159).

The liver architecture is abnormal, with increased fat deposition. The brain of a neonate who succumbed to the disease was edematous, with poor demarcation of gray and white matter. The cortical layers were poorly developed, and myelination was defective with vacuolated myelin sheaths and cystic degeneration of white matter (160). An older patient had atypical astrocytes similar to the Alzheimer II cells seen in Wilson disease and in severe chronic liver disease (161).

As ascertained by newborn screening, the incidence of argininosuccinic aciduria is 1 in 70,000 in Massachusetts and 1 in 91,000 in Austria (162). Three distinct clinical forms have been recognized, each resulting from a different genetic mutation (163).

The most severe entity is the neonatal form. Infants feed poorly, become lethargic, develop seizures, and generally die within 2 weeks (160,164). In a second form, progression is less rapid, but similar symptoms appear in early infancy. In the majority of patients, including those described by Allan and coworkers (156), the presenting symptoms are mental retardation, recurrent generalized convulsions, poorly pigmented, brittle hair (trichorrhexis nodosa), ataxia, and hepatomegaly (165). Some patients have been seizure free, however, and have presented with little more than learning difficulties, and others (approximately 20% of all affected children) have normal intelligence without treatment (166).

The presence of elevated blood ammonia should suggest a disorder in the urea cycle. Initial evaluation of such a child should include routine blood chemistries, plasma lactate levels, liver function tests, quantitative assay of plasma amino acids, and assay of urine for organic acids and orotic acid (153). The specific diagnosis of argininosuccinic aciduria can be made by a significant elevation of plasma citrulline and the presence of large quantities of plasma, urinary, and CSF argininosuccinic acid. In some instances, fasting blood ammonia level can be normal or only slightly elevated, but marked elevations occur after protein loading. Increased excretion of orotic acid is seen in all urea cycle defects, with the exception of CPS deficiency (167).

Hyperammonemic coma in the neonate caused by any of the urea cycle defects requires prompt intervention. In essence, treatment consists of detoxification and removal of the excess ammonia and reduction in the formation of ammonia. Quantitative amino acid chromatography should be performed on an emergency basis, and the infant should be given a high dose of intravenous glucose with insulin to suppress protein catabolism. The elevated ammonia levels can be reduced by hemodialysis if available or by peritoneal dialysis (168). Details of treatment are presented by Brusilow and Horwich (166) and Batshaw and colleagues (169). Treatment for increased intracranial pressure, which frequently accompanies neonatal hyperammonemia, is symptomatic.

The long-term management of an infant who suffers a urea cycle defect is directed toward lowering blood ammonia levels and maintaining them as close to normal as possible. This is accomplished by providing the infant with alternative pathways for waste nitrogen synthesis and excretion. Infants with argininosuccinic aciduria are placed on a protein-restricted diet (1.2 to 2.0 g/kg per
day), which is supplemented with L-arginine (0.4 to 0.7 g/kg per day), which promotes the synthesis of citrulline and argininosuccinate as waste nitrogen products, and citrate, which improves weight gain and reduces hepatomegaly (166,169). Sodium phenylbutyrate is added to divert ammonia from the urea cycle. A number of centers are now recommending the use of liver transplantation for argininosuccinic aciduria (170).

Episodes of hyperammonemia are generally triggered by intercurrent infections. Prevention of a rapid progression to death requires hospitalization and the use of intravenous therapy (166).

On this regimen, some children with argininosuccinic aciduria do well. Reduction of blood ammonia levels is accompanied by improved growth, reduction in liver size, cessation of seizures, and, in some patients, normal hair. Intellectual function is significantly impaired, however. In the experience of Batshaw and coworkers, all infants with the severe, neonatal form of the disease survived, although those who have been followed the longest have shown a significant lowering of their IQ (169). In the French long-term follow-up of 15 patients with argininosuccinic aciduria, none were doing well (171). Children with the late-onset variant fare much better, and, with therapy, achieve normal development (165). As a rule, the individual’s ultimate IQ is a function of the severity and duration of hyperammonemic coma, and children who have not recovered from coma within 5 days do poorly (172). Valproic acid cannot be used in the treatment of seizures associated with this and with the other urea cycle defects because it induces severe hyperammonemia at even low doses (173).

The gene coding for argininosuccinic acid synthetase has been cloned. It is carried on chromosome 9 (175). At least 50 different genetic mutations have been recorded for infants with neonatal citrullinemia (176).

As a result of the enzymatic defect, the concentration of citrulline in urine, serum, and CSF is markedly increased, and administration of a protein meal results in a dramatic increase of blood ammonia and urinary orotic acid. Blood and urinary urea values are normal, indicating that urea production is not completely blocked. CT and MRI studies performed on patients with the neonatal form of citrullinemia show lesions in the thalami, basal ganglia, cortex, and subcortical white matter. Diffusion-weighted MR images indicate the presence of cytotoxic edema. Follow-up studies reveal subcortical cysts, ulegyria, and atrophy (177,178).

In Western countries, the most common presentation is in the neonatal period with lethargy, hypotonia, and seizures (179). In other instances, the disease is less severe, even though recurrent bouts of vomiting, ataxia, and seizures can start in infancy. A third form presents with mental retardation. Completely asymptomatic individuals also have been encountered (174,179).

Treatment for citrullinemia is similar to treatment for argininosuccinic aciduria, except that for long-term therapy, the low-protein diet is supplemented with arginine and sodium phenylbutyrate (166,169).

Citrullinuria in the absence of citrullinemia has been seen in patients with cystinuria. In this instance, citrulline is derived from arginine, which is poorly absorbed from the intestine (180).

Late-onset citrullinemia with loss of enzymatic activity in liver, but not in kidney or fibroblasts, is seen predominantly in Japan, where it constitutes the most common form of citrullinemia (181). It presents with cyclical changes in behavior, dysarthria, and motor weakness. It is due to mutations in citrin, a mitochondrial aspartate glutamate carrier (182,183).

Another cause for intermittent hyperammonemia is ornithinemia. Clinically and biochemically this is a heterogeneous entity, At least two conditions have been delineated.

Transient hyperammonemia with consequent profound neurologic depression can be encountered in asphyxiated infants or with significant dehydration (217,218). This state should be differentiated not only from the various urea cycle defects, but also from the various organic acidemias, notably methylmalonic acidemia and propionic acidemia, which can induce hyperammonemia (219). In these conditions, the accumulation of organic acids inhibits the formation of N-acetylglutamine, the activator of mitochondrial CPS, and the activities of all five enzymes of the urea cycle are depressed. On a clinical basis, the organic acidemias can be distinguished from urea cycle defects in that infants with a urea cycle defect are asymptomatic for the first 24 hours of life and only rarely develop coma before 72 hours. Additionally, they demonstrate tachypnea rather than a respiratory distress syndrome. In contrast to the distressed neonates with hyperammonemia, infants with the various organic acidemias demonstrate ketonuria or ketonemia as well as acidosis (220).

Asymptomatic hyperammonemia is relatively common in low-birth-weight neonates. It probably is caused by shunting of blood away from the portal circulation of the liver into the systemic circulation.

Mild hyperammonemia can also be seen in associated with hypoglycemia and hyperinsulinism due to mutations in the glutamate dehydrogenase gene (221).

The increased excretion of homocystine is a manifestation of several inborn errors of methionine metabolism. The most common of these errors is marked by multiple thromboembolic episodes, ectopia lentis, and mental retardation. Although discovered as late as 1962 by Field and reported subsequently by Carson and coworkers (222), the prevalence of homocystinuria varies considerably from one country to another, ranging from 1 in 65,000 in Ireland to approximately 1 in 335,000 worldwide (223). The gene for this autosomal recessive condition has been cloned; it is localized to the long arm of chromosome 21 (21q22.3).

Several other conditions are marked by elevated plasma methionine levels. The most common of these is a transitory methioninemia, seen in infants, many of who are premature and receiving a high-protein diet (at least 7 g/kg per day). It is likely that the biochemical abnormality is caused by delayed maturation of one or more of the enzymes of methionine metabolism.

Methioninemia, with or without tyrosinemia, accompanied by hepatorenal disease (tyrosinemia I) is considered in the section on tyrosinosis and tyrosinemia (see Table 1.9).

Persistent methioninemia associated with a deficiency of hepatic methionine adenosyltransferase is a benign metabolic variant unaccompanied by neurologic symptoms or impairment of cognition (246).

The symptoms are the result of an extensive disturbance in sodium-dependent transport of neutral amino acids across the membrane of the brush border of the small intestine and the proximal renal tubular epithelium. Four main biochemical abnormalities exist: a renal aminoaciduria, increased excretion of indican, an abnormally high output of nonhydroxylated indole metabolites, and increased fecal amino acids. These deficits are discussed in detail by Milne and colleagues (265) and Scriver (266).

Pathologic changes in the brain are nonspecific and are limited to neuronal degeneration and dysmyelination (267).

The incidence of the biochemical lesion responsible for Hartnup disease is 1 in 18,000 in Massachusetts, 1 in 70,000 in Vienna, and 1 in 33,000 in New South Wales, Australia (268). Clinical manifestations of Hartnup disease are the consequence of several factors. Polygenic inheritance is the major determinant for plasma amino acid levels, and symptoms are seen only in patients with the lowest amino acid concentrations. Because protein malnutrition further lowers amino acid levels, the disease
itself, as distinguished from its biochemical defect, is seen mainly in malnourished children. Whenever dietary intake is satisfactory, neither neurologic nor dermatologic signs appear (268). In addition, no difference exists in rate of growth or IQ scores between groups with Hartnup disease and control groups. In the series of Scriver and coworkers, 90% of Hartnup patients had normal development (269). However, low academic performance and impaired growth were seen in those patients with Hartnup disease who, for genetic reasons, tended to have the lowest plasma amino acid levels (269).

When present, symptoms are intermittent and variable, and tend to improve with increasing age. A characteristic red, scaly rash appears on the exposed areas of the face, neck, and extensor surfaces of the extremities. This rash resembles the dermatitis of pellagra and, like it, is aggravated by sunlight. Cerebral symptoms can precede the rash for several years. They include intermittent personality changes, psychoses, migraine-like headaches, photophobia, and bouts of cerebellar ataxia. Changes in hair texture also have been observed. The four children of the original Hartnup family underwent progressive mental retardation, but this is not invariable. Renal and intestinal transport is impaired in 80% of patients and renal transport alone in 20% (269). The MRI is nonspecific; it demonstrates delayed myelination (270).

Hartnup disease should be considered in patients with intermittent cerebral symptoms, even without skin involvement.

Numerous metabolic disorders with a partial enzymatic defect produce intermittent cerebellar ataxia. These include MSUD, lactic acidosis, pyruvate dehydrogenase deficiency, and some of the diseases caused by defects in the urea cycle. Additionally, episodic ataxia should be considered. This condition is caused by mutations in the calcium-channel voltage-dependent, P/Q type, alpha 1A subunit gene (CACNA1A), which is highly expressed in the cerebellum. Another rare condition to be considered in the differential diagnosis of Hartnup disease is hypertryptophanemia (see Table 1.12) and a disorder in kynurenine hydroxylation (271). Chromatography of urine for amino acids and indolic substances in the presence of a normal serum pattern is diagnostic for Hartnup disease. The absence of increased proline and hydroxyproline in the urine distinguishes Hartnup from generalized aminoaciduria that can be the result of many other disorders.

The similarity of Hartnup disease to pellagra has prompted treatment with nicotinic acid (25 mg/day). Tryptophan ethylester also has been effective (272). However, the tendency for symptoms to remit spontaneously and for general improvement to occur with improved dietary intake and advancing age makes such therapy difficult to evaluate.

In 1917, Göppert demonstrated that galactosemic children excreted galactose after the ingestion of lactose (milk) and galactose (293). In 1956, Schwarz and associates found that administration of galactose to affected children gave rise to an accumulation of galactose-1-phosphate (294). This was confirmed by Kalckar and his group, who were able to demonstrate a deficiency in galactose-1-phosphate uridyltransferase (GALT), the enzyme that catalyzes the conversion of galactose-1-phosphate into galactose uridine diphosphate (UDP-galactose) (Fig. 1.8) (295). The gene for galactosemia has been mapped to the small arm of chromosome 9 (9p13). It has been cloned and sequenced, and numerous mutations have been identified (296,297). Two point mutations (Q188R and K285N) account for 69% to 80% of galactosemia in whites and result in a complete enzyme deficiency; most of the remaining mutations result in detectable amounts of enzyme activity (296,297). In African-American patients, the most common mutation (S135L) accounts for 45% of the mutant alleles. Patients homozygous for the S135L allele have residual red cell GALT activity and a milder clinical course (298). Compound heterozygotes of a classic galactosemia allele and the Los Angeles or Duarte variant are asymptomatic.

In classic galactosemia, the metabolic block is essentially complete. Lactose of human or cow’s milk is hydrolyzed to galactose and glucose, the latter being handled in a normal manner. The metabolism of galactose, however, stops after the sugar is phosphorylated to galactose-1-phosphate. This phosphate ester accumulates in erythrocytes, lens, liver, and kidney (299). Galactitol, the alcohol of galactose, also is found in the lens, brain, and urine of galactosemic individuals (300), and can be demonstrated in brain by MRS (303). Berry and coworkers postulated that galactitol induces cerebral edema that is occasionally present in neonates (301). Factors responsible for mental retardation can include a deficiency of uridine diphosphate galactose, a reduction of glycolytic intermediates, and a loss of adenosine triphosphate (ATP) (300).

Administration of galactose to affected infants results in marked hypoglycemia. This has been explained by an increased insulin release, prompted by the large amounts of circulating reducing substance, or by assuming interference by galactose-1-phosphate with normal glycogen
breakdown. The peripheral hypoglycemia is enhanced by competition between glucose and galactose at the level of the hexose transport across the blood–brain barrier. As a consequence, the brain in galactosemic patients is in a constant hypoglycemic environment.

Intolerance to galactose decreases with increasing age. In part, this intolerance might be a result of the decreasing importance of milk as a food item. In patients with classic galactosemia who have been tested repeatedly, no increase in erythrocyte transferase activity has been found (302).

The main pathologic lesions are found in the liver and brain. In the liver, several stages are recognized. Initially one sees a severe, diffuse, fatty metamorphosis. The hepatic cells are filled with large, pale, fat-containing vacuoles (303). If the disease remains untreated, the liver cell cords are transformed into pseudoglandular structures. The final stage is pseudolobular cirrhosis. Cerebral alterations are nonspecific. Edema, fibrous gliosis of white matter and marked loss of cortical neurons and Purkinje cells are the most prominent findings (304).

Infants with galactosemia appear healthy at birth, although their cord blood can already contain abnormally high concentrations of galactose-1-phosphate, and a few already have cataracts and hepatic cirrhosis, probably as a consequence of intrauterine galactosemia (305). In severe cases, symptoms develop during the first week of life. These include vomiting, diarrhea, listlessness, and failure to gain weight. Increased intracranial pressure due to cytotoxic cerebral edema can be a presenting sign (306). Infants are jaundiced. This may represent a persistence of neonatal jaundice or can appear at age 3 to 5 days (307). On a normal diet, hypoglycemia is not common. By age 2 weeks, hepatosplenomegaly and lenticular opacifications are easily detectable. The cataracts can be cortical or nuclear and can be present at birth (300). The most frequently observed opacity is a central refractile ring (308). The infants are hypotonic and often have lost the Moro reflex. Sepsis caused by Escherichia coli occurs with high frequency and is responsible for the majority of deaths during the neonatal period (309). Other secondary effects of deranged galactose metabolism include ovarian failure or atrophy and testicular atrophy (310). The MRI commonly shows multiple areas of increased signal in white matter, predominantly in the periventricular region (311).

If galactosemia goes untreated, growth failure becomes severe and the infant develops the usual signs of progressive hepatic cirrhosis. In some infants, the disease can be less severe and does not manifest until age 3 to 6 months, at which time the presenting symptoms are delayed physical and mental development. By then, cataracts can be well established and the cirrhosis far advanced.

In another group of galactosemic individuals, the diagnosis is not made until patients are several years old, often on evaluation for mental retardation. They might not have cataracts or albuminuria. Intellectual retardation is not consistent in untreated galactosemic children. When present, it is moderate; IQs range between 50 and 70. Asymptomatic homozygotes also have been detected. Most of these have some residual transferase activity.

The enzyme defect is best documented by measuring the erythrocyte GALT activity. Several methods are available and have been be used for statewide and nationwide screening. In the Austrian screening program reviewed by Item and coworkers, only 1 in 190 infants who tested positive initially actually had galactosemia (297). Some of these were galactosemia carriers or Duarte/galactosemia compound heterozygotes.

Galactosuria, usually in combination with glucosuria or fructosuria, is seen in severe hepatic disorders of the neonatal period (e.g., neonatal hepatitis, tyrosinosis, congenital atresia of the bile ducts). Families in which several members are mentally defective and have congenital cataracts without an abnormality in galactose metabolism have been described by Franceschetti and others (312).

The antenatal diagnosis of galactosemia can be made by assay of GALT activity on cultured amniocytes or chorionic villi (315).

When milk is withdrawn and lactose-free products such as Nutramigen or Prosobee are substituted, gastrointestinal symptoms are rapidly relieved and normal growth resumes. The progression of cirrhosis is arrested, and in 35% of patients, the cataracts disappear (307). Because infants have developed hypoglycemia when first placed on a galactose-free diet, it might be useful to add some glucose to the formula.

The propensity of galactosemic neonates for E. coli sepsis requires securing cultures of blood, urine, and CSF and treating against this organism until the test results are negative (309). In the larger series of Waggoner and coworkers, sepsis was suspected in 30% of neonates and confirmed in 10% (314).

Recommendations for the management of infants and children with galactosemia have been published by Walter and coworkers (315). Maintenance of the galactose-free diet and avoidance of milk and milk products is recommended until at least after puberty. In several children who returned to a milk-containing diet before puberty, cataracts flared up. Even after that age, some continue to be sensitive to milk products and prefer to avoid them. Intermittent monitoring of erythrocyte galactose-1-phosphate levels has been suggested. Even
children with well-controlled galactosemia have elevated galactose-1-phosphate levels. Endogenous formation of galactose-1-phosphate from glucose-1-phosphate by way of the epimerase reaction (see Fig. 1.8) is believed to be responsible.

The long-term outlook for galactosemic patients is not as good as was initially believed, even when the diet is carefully monitored. A number of cases of progressive cerebellar ataxia and extrapyramidal movement disorders have been reported (316,317). In the series of Waggoner and colleagues, cerebellar deficits were seen in 18% (314); in the series of Kaufman and colleagues, the incidence was 27% (317). The cause for this syndrome is unclear, and MRI studies on patients with this syndrome do not differ from those without it (317). Rogers and Segal postulated that the syndrome results from an endogenous product of galactose-1-phosphate from UDP-glucose via the epimerase reaction (318). Another possibility is that a deficiency of UDP-galactose could limit the formation of cerebral glycoproteins and galactolipids. Administration of uridine, which has been suggested to prevent these complications, has not been effective (319).

Cognitive deficits are common. In 70% of galactosemic children treated from birth, the IQ was 90 or higher; however, approximately 50% of the youngsters had significant visual and perceptual deficits, and 33% had EEG abnormalities (320). Studies confirm that most patients have cognitive deficits in one or more areas. Verbal dyspraxia occurs in some 62%. Patients are unable to program their speech musculature and also show frequent disturbances in speech rhythm. Receptive language is normal (317,321). In addition, there appears to be a progressive decline in IQ with age (314,317). Neither IQ scores nor the presence of the dyspractic speech disorder are highly related with the age at which therapy is initiated or quality of control (314). Instead, cognitive and language deficits are likely to result from the in utero formation of potentially neurotoxic galactose-1-phosphate and the continued generation of galactose from glucose via UDP-galactose-4-epimerase (see Fig. 1.8) (300,322).

Two other defects of galactose metabolism have been recognized. A deficiency of galactokinase (see Fig. 1.8) is the more common (OMIM 230200). Cataracts are present in most patients and pseudotumor cerebri has been seen occasionally. The outlook in terms of mental function is better than for patients with galactosemia, and most appear to be normal (323). A very rare disorder, generalized deficiency of epimerase (OMIM 230350), the enzyme that converts UDP-glucose to UDP-galactose (see Fig. 1.8), can result in galactosuria, failure to thrive, sensorineural deafness, dysmorphic features, and mental retardation (324).

This is a recessive disorder with an incidence of approximately 1 in 30,000. It is caused by a defect in the gene for glutaryl-CoA dehydrogenase. Biochemically, the condition is marked by the excretion of large amounts of glutaric, 3-hydroxyglutaric, and glutaconic acids. The clinical picture is protean, and the disorder is frequently undiagnosed or misdiagnosed. In approximately 20% of patients, glutaric aciduria I takes the form of a neurodegenerative condition commencing during the latter part of the first year of life and characterized by hypotonia, dystonia, choreoathetosis, and seizures (377). In most of the remaining patients, development is normal for as long as 2 years of age, and then, after what appears to be an infectious, an encephalitic, or a Reye syndrome–like illness or following routine immunization, neurologic deterioration occurs (378,379). In yet other cases, the clinical picture
resembles extrapyramidal cerebral palsy (380). Rare cases remain asymptomatic. Macrocephaly is noted in 70% of cases (381). Lack of appetite, sleeplessness, and profuse sweating also is noted, as is hypoglycemia (382,383). No molecular basis for the clinical variability exists, and the severity of the clinical phenotype seems to be closely linked to the development of encephalopathic crises rather than to residual enzyme activity or genotype (384).

Neuroimaging shows frontotemporal cortical atrophy giving what has been termed a bat wing appearance. This is often accompanied by increased signal in the basal ganglia on T2-weighted images and caudate atrophy. Bilateral subdural hematomas have also been described (Fig. 1.9). When these are accompanied by retinal hemorrhages, an erroneous diagnosis of child abuse frequently is made (390).

Neuropathology shows temporal and frontal lobe hypoplasia, degeneration of the putamen and globus pallidus, mild status spongiosus of white matter, and heterotopic neurons in cerebellum (385). It is not clear why so much of the damage is localized to the basal ganglia. Kolker and coworkers postulated that 3-hydroxyglutaric acid, which is structurally related to glutamic acid, induces excitotoxic cell damage, and that in addition 3-hydroxyglutaric acid and glutaric acid modulate glutamatergic and gamma-aminobutyric acid (GABA)-ergic neurotransmission. Secondary amplification loops could also potentiate the neurotoxic properties of these organic acids (386).

A low-protein, high-calorie diet supplemented with a formula lacking lysine and tryptophan and containing carnitine has sometimes prevented further deterioration, but in the Scandinavian series of Kyllerman and colleagues almost all patients were left with a severe dystonic-dyskinetic disorder (387). The experience of Hoffmann and coworkers was similar (381). Strauss and colleagues, who worked with the disease in the Pennsylvania Amish population, however, found that good dietary care can reduce the incidence of basal ganglia injury to 35% (388).

In this condition the defect has been localized to one of three genes involved in the mitochondrial β-oxidation of fatty acids: those coding for the α and β subunits of the electron transfer flavoprotein (ETF) and that coding for the electron transfer flavoprotein dehydrogenase (389,390). The clinical picture of GA II due to the different defects appears to be nondistinguishable; each defect can lead to a range of mild or severe cases, depending presumably on the location and nature of the intragenic lesion, that is, mutation, in each case. As a consequence of the enzyme deficiency there is increased excretion not only of glutaric acid, but also of other organic acids, including lactic, ethylmalonic, isobutyric, and isovaleric acids.

The heterogeneous clinical features of patients with MADD fall into three classes (391): a neonatal-onset form with congenital anomalies (type I), a neonatal-onset form without congenital anomalies (type II), and a late-onset form (type III).

Glutaric aciduria II can present in the neonatal period with an overwhelming and generally fatal metabolic acidosis coupled with hypoglycemia, acidemia, and a cardiomyopathy. Affected infants can have an odor of sweaty feet. As a rule, there is good correlation between the severity of the metabolic block, rather than its location and the severity of the disease, with null mutations producing the development of congenital anomalies and the presence of a minute amount of enzymatic activity allowing the development of type II disease (392).

Dysmorphic features are prominent in approximately one-half of cases (393). They include macrocephaly, a large anterior fontanel, a high forehead, a flat nasal bridge, and malformed ears. This appearance is reminiscent of Zellweger syndrome (Fig. 1.10). Neuroimaging discloses agenesis of the cerebellar vermis and hypoplastic temporal lobes (394). The neonatal-onset forms are usually fatal and are characterized by severe nonketotic hypoglycemia, metabolic acidosis, multisystem involvement, and excretion of large amounts of fatty acid- and amino acid-derived metabolites.

Standard treatment consists of a high-carbohydrate, low-fat and protein diet supplemented with carnitine. Van Hove and coworkers suggested that this condition be treated with D,L-3-hydroxybutyrate (430 to 700 mg/kg
per day). In their experience this treatment resulted in improvement of neurologic function and cardiac contractility (395).

Symptoms and age at presentation of late-onset MADD (type III) are highly variable and characterized by recurrent episodes of lethargy, vomiting, hypoglycemia, metabolic acidosis, and hepatomegaly often preceded by metabolic stress. Muscle involvement in the form of pain, weakness, and lipid storage myopathy also occurs. The organic aciduria in patients with the late-onset form of MADD is often intermittent and only evident during periods of illness or catabolic stress. Other cases present in early childhood with progressive spastic ataxia and high signal intensity on T2-weighted MRI in supratentorial white matter, a picture mimicking a leukodystrophy (398).

The neuropathologic picture is marked by diffuse gliosis of cerebrum, brainstem, and cerebellum, with foci of leukomalacia and striatal degeneration (393,397).

Some infants with glutaric aciduria II respond dramatically to riboflavin (100 mg three times a day). This riboflavin-responsive multiple acyl-CoA dehydrogenase deficiency may be identical to ethylmalonic-adipic aciduria (396,398,399).

This condition is the result of a defect of peroxisomal glutaryl CoA oxidase. It results in a persistent isolated glutaric acid excretion, but the phenotype of this disorder is unknown, and it may well represent a benign inborn error (400).

The other, rarer organic acidurias are summarized in Table 1.15.

The other disorders of fatty acid β-oxidation are much less common. They are summarized in Table 1.14. In VLCAD the defect is at the first step of the fatty acid β-oxidation cycle. Clinically, failure of long-chain fatty acid β-oxidation leads to hypoketotic hypoglycemia associated with coma, liver dysfunction, skeletal myopathy, and hypertrophic cardiomyopathy. Patients are unable to metabolize fatty acids with 12 to 18 carbons (420,421,422,423). Three clinical forms have been recognized: a severe form with early onset and a high incidence of cardiomyopathy; a milder, childhood-onset form; and an adult form with rhabdomyolysis and myoglobinuria (422,423,424). The clinical picture appears to correlate with the amount of residual enzyme activity (417). VLCAD can be effectively treated with avoidance of fasting and a diet low in long-chain fats supplemented with medium-chain triglyceride oil and carnitine. Uncooked cornstarch at night can be added to the regimen (425).

Two groups of enzymes are involved in the degradation of glycogen. Phosphorylase initiates one set of breakdown reactions, cleaving glycogen to limit dextrin, which then is acted on by a debrancher enzyme (oligo-1,4-glucano-transferase and amyl-1,6-glucosidase) to yield a straight-chain polyglucosan, which is cleaved to the individual glucose units by phosphorylase. A second set of enzymes includes α-amylase, which cleaves glycogen to a series of oligosaccharides, and two α-1,4-glucosidases, which cleave the α-1,4 bonds of glycogen. There are two of these terminal glucosidases. One, located in the microsomal fraction, has a neutral pH optimum (neutral maltase); the other, found in lysosomes, has its maximum activity at an acid pH (3.5) (acid α-glucosidase). The gene for the latter enzyme has been mapped to the long arm of chromosome 17 (17q23) and has been cloned.

In type II glycogenosis, deficiency of lysosomal acid α-glucosidase leads to accumulation of lysosomal glycogen predominantly in liver, heart, and skeletal muscle (468). Like every other lysosomal protein, α-glucosidase is synthesized in a precursor form on membrane-bound polysomes and is sequestered in the lumen of the rough endoplasmic reticulum as a larger glycosylated precursor. Whereas the primary translational product of α-glucosidase mRNA is 100 kd, it becomes glycosylated to yield a 110-kd precursor, which in turn undergoes a posttranslational modification by phosphorylation to yield the mannose-6-phosphate recognition marker (469). Phosphorylation is an important step because formation of the marker distinguishes the lysosomal enzymes from
secretory proteins and allows the lysosomal enzymes to be channeled to the lysosomes, to which they become attached by a membrane-bound mannose-phosphate–specific receptor. Additionally, the 110-kd precursor undergoes proteolytic processing to yield the two 76-kd and one 70-kd mature lysosomal enzyme polypeptides. With such a complex processing system, it is not surprising to encounter extensive clinical heterogeneity in glycogenosis II. Considerable genetic heterogeneity exists, and a variety of nonsense, frameshift, and missense mutations have been described. These result in partial or complete loss of enzyme activity. As a rule, the amount of residual enzyme activity correlates fairly well with the severity of the disease. Two relatively common mutations are responsible for a large proportion of the infantile form of Pompe disease. In whites, one of the more common mutations is a deletion of exon 18, which results in total loss of enzyme activity and the clinical manifestations of infantile glycogen storage disease (470,471).

Glycogen can be deposited in virtually every tissue. The heart is globular, the enlargement being symmetric and primarily ventricular. In the infantile form, massive glycogen deposition occurs within the muscle fibers. In cross section, these appear with a central, clear area, giving a lacunate appearance. Glycogen also is deposited in striated muscle (particularly the tongue), in smooth muscle, and in the Schwann cells of peripheral nerves, kidney, and liver (Fig. 1.11). Ultrastructural studies of muscle show glycogen to be present in lysosomal sacs, where it occurs in conjunction with cytoplasmic degradation products. Additionally, it occurs free in cytoplasm (472). In the CNS, glycogen accumulates within neurons and in the extracellular substance (473). The anterior horn cells are affected predominantly, although deposits are seen in all parts of the neuraxis, including the cerebral cortex (474). Chemical analysis indicates an excess of glycogen in cerebral cortex and white matter and a deficiency in total phospholipid, cholesterol, and cerebroside. No evidence exists for primary demyelination.

Pompe disease can take one of three forms. In the classic, infantile form, the first symptoms usually appear by
the second month of life. They include difficulty in feeding, with dyspnea and exhaustion interfering with sucking. Gradually, muscular weakness and impaired cardiac function become apparent. Marked cardiac enlargement is present at an early age. The heart appears globular on radiographic examination; murmurs are usually absent, but the heart tones have a poor quality and a gallop rhythm is often audible. The electrocardiogram shows characteristic high-voltage QRS complexes. Affected infants have poor muscle tone and few spontaneous movements, although the deep tendon reflexes are often intact. Skeletal muscles are often enlarged and gradually acquire a peculiar rubbery consistency. Electromyography shows lower motor neuron degeneration (475). Convulsions, intellectual impairment, and coma also have been observed. The liver, although usually not enlarged, is abnormally firm and easily palpable. Splenomegaly is rare. Infants are prone to intercurrent infections, particularly pneumonia, and usually die of these or bulbar paralysis by 1 year of age. In most patients having this clinical form, lysosomal α-glucosidase is catalytically inactive, although the protein is present in some families. The most common form of this variant, termed Antopol disease, is marked by severe cardiomyopathy, a mild myopathy, and mental retardation (476). Untreated, only 8% of all reported patients have survived beyond the first year of life (477).

α-Glucosidase deficiency also has been seen in older children and adults (juvenile and adult forms, respectively) (478). In these patients, organomegaly is absent, and muscle weakness is often slowly progressive or nonprogressive with maximum involvement of the proximal musculature of the lower extremities (479). Pharyngeal muscles can be involved as a consequence of glycogen storage in brainstem neurons (480). In most of the slowly progressive cases, residual lysosomal α-glucosidase activity is more than 10%. In others, the enzyme activity is undetectable, but the 110-kd precursor is present. Other individuals appear to be compound heterozygotes for different mutant alleles (478).

When cardiac symptoms predominate, a differentiation from other causes of cardiomegaly and congestive failure in the absence of significant murmurs is required. These other causes include endocardial fibroelastosis, acute interstitial myocarditis, and aberrant coronary artery. When muscular weakness predominates, infantile spinal muscular atrophy (Werdnig-Hoffmann disease), the muscular dystrophies, myasthenia gravis, and hypotonic cerebral palsy must be considered (see Chapter 16). Cardiac involvement is generally late in Werdnig-Hoffmann disease and in the muscular dystrophies. In contrast to glycogen storage disease, the intellectual deficit in hypotonic cerebral palsy is usually severe and begins early. A muscle biopsy may be required to confirm the diagnosis. Blood chemistry, including fasting blood sugars and glucose tolerance tests, is normal.

α-Glucosidase activity of muscle and cultured fibroblasts is markedly diminished, and this assay serves as an excellent diagnostic aid (481). Examination of lymphocytes by electron microscopy frequently is successful in demonstrating and identifying the storage material. α-Glucosidase activity also can be shown in fibroblasts grown from normal amniotic fluid cells. This method can also be used for the antepartum diagnosis of Pompe disease and for the detection of heterozygotes.

Several approaches to the treatment of glycogenosis II, as well as the other lysosomal defects, have been proposed. Enzyme replacement therapy, using twice-weekly infusions of recombinant human enzyme from rabbit milk or from genetically engineered Chinese hamster ovary cells overproducing acid α-glucosidase, has been used for the infantile and the late-onset forms of Pompe disease (482,483,484). The results have been encouraging in a few cases with the infantile form when treatment is started before irreversible damage has occurred (482). The cardiac muscle universally responds, whereas the response of skeletal muscle is variable. Because the enzyme does not cross the blood–brain barrier, it remains to be seen whether there will be late-onset complications in treated infantile Pompe patients. In the juvenile form of the disease enzyme replacement therapy has also resulted in some improvement (483). Adenovirus-mediated gene transfer holds some promise in delivering α-glucosidase to muscle (485). The problems inherent in gene delivery in Pompe disease are discussed by Ding and coworkers (486), and gene delivery to the brain is discussed by Kennedy (487).

Allogeneic bone marrow transplantation offers more promise for the treatment of some lysosomal storage diseases. In glycogenosis II, however, no beneficial result has been reported, despite successful engraftment. Liver transplantation has been used with some effect in types I, III, and IV glycogen storage disease (488). After transplant, the cells of the host organs become mixed with cells of the donor genome that migrate from the allograft into the tissues of the recipient and serve as enzyme carriers. This observation explains why some patients with such lysosomal storage diseases as type I Gaucher disease, Niemann-Pick disease, and Wolman disease receive more benefit from liver transplants than simply improved hepatic functions.

The principal biochemical disturbance in the various mucopolysaccharidoses involves the catabolism of MPS. The chemistry of the MPS, also known as glycosaminoglycans, has been reviewed by Alberts and colleagues (495) and more extensively by Kjellen and Lindahl (496). These substances occur as large polymers having a protein core and multiple carbohydrate branches. Chondroitin-4-sulfate is found in cornea, bone, and cartilage. It consists of alternating units of D-glucuronic acid and sulfated N-acetylgalactosamine (Fig. 1.12).

Dermatan sulfate is a normal minor constituent of connective tissue and a major component of skin. It differs structurally from chondroitin-4-sulfate in that the uronic acid is principally L-iduronic rather than glucuronic acid (Fig. 1.12).

Heparan sulfate consists of alternating units of uronic acid and glucosamine. The former may be L-iduronic or D-glucuronic acid. Glucosamine can be acetylated on the amino nitrogen as well as sulfated on the amino nitrogen and hydroxyl group (Fig. 1.12). Generally, the MPS chains are linked to the serine of the protein core by means of a xylose-galactose-galactose-glucuronic acid-N-acetylhexosamine bridge.

The pathways of MPS biosynthesis are well established. The first step is the formation of a protein acceptor. Hexosamine and uronic acid moieties are then attached to the protein, one sugar at a time, starting with xylose. Sulfation occurs after completion of polymerization. The sulfate groups are introduced through the intermediary of an active sulfate. This is a labile nucleotide, identified as adenosine-3′-phosphate-5′-sulfatophosphate.

In the mucopolysaccharidoses, degradation of MPS is impaired because of a defect in one of several lysosomal hydrolases. This results in the accumulation of incompletely degraded molecules within lysosomes and the excretion of MPS fragments. The initial steps of MPS degradation (cleavage from the protein core) are intact in the mucopolysaccharidoses. In tissue where amounts of MPS are pathologic, such as liver, spleen, and urine, the protein core is completely lacking or represented only by a few amino acids.

In all of these diseases, therefore, the metabolic defect is located along the further degradation of the MPS, and in all but one, it involves the breakdown of dermatan sulfate and heparan sulfate (Fig. 1.12). As a result, large amounts (75 to 100 mg per 24 hours) of fragmented chains of these MPSs are excreted by individuals afflicted with the two major genetic entities: the autosomally transmitted Hurler syndrome (MPS IH; see Table 1.18) and the rarer X-linked Hunter syndrome (MPS II). These values compare with a normal excretion of 5 to 25 mg per 24 hours for all MPSs. Normal MPS excretion is distributed between chondroitin-4-sulfate (31%), chondroitin-6-sulfate (34%), chondroitin (25%), and heparan sulfate (8%) (497).

The biochemical lesions in the various mucopolysaccharidoses have been reviewed by Neufeld and Muenzer (498).

In the Hurler syndromes (MPS I), the activity of α-L-iduronidase is deficient (Fig. 1.12, step 1) (434). The iduronic acid moiety is found in dermatan and heparan sulfates (Fig. 1.12); therefore, the metabolism of both MPSs is defective. The gene for α-L-iduronidase has been mapped to chromosome 4 and has been cloned. Several hundred mutations have been described, with the frequency of each mutation differing from one genetic stock to the next. As a consequence of the genetic variability, a wide range of clinical phenotypes exists, from the most severe form, Hurler syndrome (IH), through an intermediate form (I H/S), to the mildest form, Scheie syndrome or MPS IS (499). Among patients of European origin, two mutations account for more than 50% of alleles (499).

In Hunter syndrome (MPS II), iduronate sulfatase activity is lost (Fig. 1.12, step 5) (500). Like L-iduronidase, this enzyme also is involved in the metabolism of dermatan and heparan sulfates. The gene for the enzyme is located on the distal portion of the long arm of the X chromosome (Xq28) in close proximity to the fragile X site. It has been cloned, and considerable genetic heterogeneity exists; in contrast to Hurler syndrome, there is a high incidence of gene deletions or rearrangements, and most families have their private mutation (500). Several phenotypic

forms of Hunter syndrome have been distinguished, and, as a rule, gene deletions result in a more severe clinical picture than point mutations. Hunter syndrome can rarely occur in female patients; in these circumstances, it results from X:autosome translocation or the unbalanced inactivation of the nonmutant chromosome (501).

In the four types of Sanfilippo syndrome (MPSs IIIA, B, C, and D), the basic defect involves the enzymes required for the specific degradation of heparan sulfate. In the A form of the disease, a lack of heparan-N-sulfatase (heparan sulfate sulfatase) occurs (Fig. 1.12, step 2), which is the enzyme that cleaves the nitrogen-bound sulfate moiety (498). In the B form, the defective enzyme is N-acetyl-α-D-glucosaminidase (Fig. 1.12, step 6) (498). In Sanfilippo syndrome, type C, the defect is localized to the transfer of an acetyl moiety from acetyl-CoA to the free amino group of glucosamine (Fig. 1.12, step 4) (498). In type D, the sulfatase for N-acetyl-glucosamine-6-sulfate is defective (Fig. 1.12, step 3) (498). The gene has been mapped to the long arm of chromosome 6 and has been cloned. Because all four of these enzymes are specific for the degradation of heparan sulfate, dermatan sulfate metabolism proceeds normally.

In Morquio syndrome (MPS IV) type A, the enzymatic lesion involves the metabolism of the structurally dissimilar keratan sulfate and chondroitin-6-sulfate. It is located at N-acetyl-galactosamine-6-sulfate sulfatase (Fig. 1.12, step 10) (498). Morquio syndrome type B, a clinically milder form, is characterized by a defect in β-galactosidase. The gene for this condition has been cloned. As a consequence of its defect, the removal of galactose residues from keratan sulfate is impaired. The defect is allelic with G_M1 gangliosidosis, a condition in which a deficiency of β-galactosidase interrupts the degeneration of keratan sulfate and G_M1 ganglioside (502). As a consequence of these defects, the urinary MPS in both forms of Morquio syndrome consists of approximately 50% keratan sulfate and lesser amounts of chondroitin-6-sulfate (see Table 1.18).

In Maroteaux-Lamy syndrome (MPS VI), the abnormality is an inability to hydrolyze the sulfate group from N-acetyl-galactosamine-4-sulfate (Fig. 1.12, step 8) (498). This reaction is involved in the metabolism of dermatan sulfate and chondroitin-4-sulfate (Fig. 1.12). The gene has been mapped to the long arm of chromosome 5 and has been cloned. Numerous mutations have been documented, and the phenotype of the condition is quite variable.

In MPS VII (Sly syndrome), a lack of β-glucuronidase (Fig. 1.12, step 7) has been demonstrated in a variety of tissues (498). The gene for this enzyme has been mapped to the long arm of chromosome 7 (7q21.11) and has been cloned. Numerous mutations have been described. As is the case for the other MPSs, these allelic forms have been postulated to explain differences in clinical manifestations in patients having the same enzymatic lesion.

Visceral alterations in Hurler syndrome are widespread and involve almost every organ. Large, vacuolated cells containing MPS can be seen in cartilage, tendons, periosteum, endocardium, and vascular walls, particularly the intima of the coronary arteries (503). Abnormalities occur in the cartilage of the bronchial tree, and the alveoli are filled with lipid-laden cells. The bone marrow is replaced in part by connective tissue that contains many vacuolated fibroblasts. The liver is unusually large, most parenchymal cells being at least double their normal size. On electron microscopy, MPSs are noted to accumulate in the lysosomes of most hepatic parenchymatous cells (504). The
Kupffer cells are swollen and contain abnormal amounts of MPS. In the spleen, the reticulum cells are larger than normal and contain the same storage material.

Changes within the CNS are widespread. The leptomeninges are edematous and thickened. The Virchow-Robin spaces are filled with MPS, and the periadventitial space in white matter is dilated and filled with viscous fluid and mononuclear cells containing MPS-positive vacuoles. The meningeal alterations produce partial obstruction of the subarachnoid spaces, which, in association with a narrowed foramen magnum, is responsible for the hydrocephalus that is often observed. The neurons are swollen and vacuolated, and their nuclei are peripherally displaced. Neuronal distention is most conspicuous in the cerebral cortex, but cells in the thalamus, brainstem, and particularly in the anterior horns of the spinal cord are involved also. In the cerebellum, the Purkinje cells contain abnormal lipid material and demonstrate a fusiform swelling of their dendrites. On electron microscopy, the storage material is in the form of membranes, zebra bodies, and, least common, lipofuscin material (505). The relationship of the pathologic appearance of neurons to the neurologic defects has been studied extensively. Because it has been best delineated in G_M2 gangliosidosis, it is discussed under that section.

From a chemical viewpoint, the material stored in the brain is of two types. Within neurons are large amounts of two gangliosides: the G_M2 ganglioside, which also is stored in the various G_M2 gangliosidoses (Tay-Sachs disease), and the hexosamine-free hematocyte (G_M3 ganglioside). The greater proportion of the stored material is within mesenchymal tissue and is in the form of dermatan and heparan sulfates (505).

A detailed review of the clinical manifestations of the various mucopolysaccharidoses can be found in the book by Beighton and McKusick (506). Hurler syndrome is inherited as an autosomal recessive disorder with an estimated incidence of 1 in 144,000 births (507). Affected children usually appear healthy at birth and, except for repeated infections, otitis in particular, remain healthy for the greater part of their first year of life. Slowed development can be the first evidence of the disorder. Bony abnormalities (dysostosis multiplex) of the upper extremities can be observed by 12 years of age and worsen thereafter. These changes are much more marked there than in the lower extremities, with the most striking being swelling of the central portion of the humeral shaft and widening of the medial end of the clavicle (Fig. 1.13), alterations that result from a thickening of the cortex and dilation of the medullary canal (508).

The typical patient is small with a large head and coarse facial features (Fig. 1.14). The eyes are widely spaced. The bridge of the nose is flat and the lips are large.
The mouth is open and an almost constant nasal obstruction or upper respiratory infection exists. Kyphosis is marked and appears early. The hands are wide and the fingers are short and stubby, with contractures that produce a claw-hand deformity. A carpal tunnel syndrome caused by entrapment of the median nerve is common, as is odontoid hypoplasia, which can lead to C1–2 vertebral displacement and quadriplegia. The abdomen protrudes, an umbilical hernia is often present, and gross hepatosplenomegaly occurs. The hair is profuse and coarse. In older children, a unique skin lesion is observed occasionally (509). It consists of an aggregation of white, nontender papules of varying size, found symmetrically around the thorax and upper extremities. Corneal opacities are invariable. In most cases, progressive spasticity and mental deterioration occur. Blindness results from a combination of corneal clouding, retinal pigmentary degeneration, and optic atrophy. Optic atrophy is the consequence of prolonged increased intracranial pressure, meningeal infiltration constricting the optic nerve, and infiltration of the optic nerve itself. Cortical blindness caused by MPS storage in neurons of the optic system also has been observed. A mixed conductive and sensorineural deafness occurs (510).

Untreated, the disease worsens relentlessly, progressing more slowly in those children whose symptoms have a somewhat later onset. Many of the slower-progressing patients suffer from the intermediate Hurler-Scheie syndrome (MPS I H/S). A curious and unique feature of this particular variant is the presence of arachnoid cysts. Spinal cord compression is not unusual; in Hurler syndrome, it generally results from dural thickening.

Radiographic abnormalities in Hurler syndrome are extensive and have been reviewed by Caffey (see Fig. 1.13) (508). They are the prototype for the other mucopolysaccharidoses. The CSF is normal. When blood smears are studied carefully, granulocytes can be seen to contain darkly azurophilic granules (Reilly granules). Some 20% to 40% of lymphocytes contain metachromatically staining cytoplasmic inclusions. These are also seen in Hunter and Sanfilippo syndromes and in the GM1 gangliosidoses. MRI not only in Hurler but also in Hunter and Sanfilippo diseases shows five types of changes: (a) Cystic, “cribriform” changes due to abnormal enlargement of the perivascular spaces with MPS-laden cells. (b) Cystic lesions of the corona radiata, periventricular white matter, corpus callosum, and. less frequently, basal ganglia. These lesions have low signal intensity of T1-weighted images and high signal intensity on T2-weighted images. They reflect MPS deposition in the perivascular spaces. In the more advanced cases, increased patchy or diffuse signal is seen in periventricular white matter on T2-weighted images. (c) Ventricular enlargement and cerebral atrophy. (d) Spinal cord compression. (e) Mega cisterna magna or posterior fossa cysts (511,512).

Death is usually a result of airway obstruction or bronchopneumonia secondary to the previously described pulmonary and bronchial alterations. Coronary artery disease and congestive heart failure caused by MPS infiltration of the heart valves also can prove fatal, even as early as 7 years of age (513).

Although classic Hurler syndrome offers little in the way of diagnostic difficulty, in many children, particularly in infants, some of the cardinal signs, such as hepatosplenomegaly, mental deficiency, bony abnormalities, or a typical facial configuration, are minimal or completely lacking (Fig. 1.15) (514). A qualitative screening test for increased output of urinary MPS usually confirms the diagnosis, whereas quantitative determinations of the various MPSs indicate the specific disorder. False-negative and false-positive test results are encountered, with the
latter being fairly common in newborns. A more definitive diagnosis requires an assay for α-L-iduronidase. If Hurler disease is suspected, the assay can be performed on lymphocytes or cultured fibroblasts (498). These assays also can serve for carrier detection or prenatal diagnosis (498).

In a number of conditions, the facial configuration seen in Hurler syndrome, the body abnormalities, and hepatosplenomegaly are unaccompanied by an abnormal MPS excretion. The first of these entities to have been defined was generalized G_M1 gangliosidosis, formerly known as pseudo-Hurler syndrome. This condition is characterized by hepatosplenomegaly, mental retardation, and bony abnormalities but a normal MPS output. The basic defect is one of ganglioside degradation (see the section on sphingolipidoses later in this chapter).

Patients presenting with dwarfism, early psychomotor retardation, unusual facies, a clear or only faintly hazy cornea, and normal MPS excretion manifest the syndrome termed mucolipidosis II, or I-cell disease. This entity is discussed with the mucolipidoses.

Other conditions to be considered in the differential diagnosis are mucolipidoses III and IV and the various disorders of glycoprotein degradation: mannosidosis, fucosidosis, sialidosis, and aspartylglycosaminuria. Finally, hypothyroidism must always be excluded in the youngster with small stature, developmental retardation, and unusual facies (see Chapter 17).

Bone marrow transplantation has dramatically improved the course of Hurler disease and its prognosis (514a). The beneficial effects result from the replacement of enzyme-deficient macrophages and microglia by marrow-derived macrophages that provide a continuous source of normal enzyme, which can enter the brain and digest stored MPS. Several hundred patients with Hurler disease have undergone bone marrow transplantation with considerable improvement of their dysmorphic features, cardiac defects, hepatosplenomegaly, and hearing. Macrocephaly tends to resolve, as does odontoid hypoplasia, but the skeletal abnormalities progress (515). In the British series of Vellodi and colleagues, the intelligence of 60% of children improved or showed no further deterioration after bone marrow transplant (516). In the American series of Guffon and colleagues, 46% of patients had normal intelligence and 15% had borderline intelligence (517). Because little improvement occurs in mental function in children who are severely retarded, Vellodi and colleagues did not recommend bone marrow transplants for those older than 2 years of age (516). Guffon and colleagues considered an initial IQ of lower than 70 to be the main criterion for excluding a patient from consideration for transplant (517). As a rule, no improvement occurs in corneal clouding, and orthopedic problems tend to persist (518). Bone marrow transplant has not improved the CNS manifestations of Hunter or Sanfillipo diseases, and the procedure is no longer performed for those conditions.

Enzyme replacement therapy is being used extensively for patients with Hurler disease as well as for some of the other MPSs. In the placebo-controlled series of Wraith and coworkers weekly infusions of 100 U/kg of α-L-iduronidase (Laronidase) significantly reduced hepatomegaly, MPS excretion, respiratory function, and sleep apnea. Growth rate and weight gain improved, and there was an increase in the range of motion of shoulder and elbow (519). Although nearly all patients developed immunoglobulin G (IgG) antibodies, these tended to fall after continued therapy as patients developed immune tolerance (520). Neurologic symptoms are not reversible with enzyme replacement therapy. Prior enzyme replacement therapy may help to improve engraftment and survival for those undergoing bone marrow transplantation (521). There is hope that intrathecal therapy may be useful for treating the brain in the future.

MPS I is frequently accompanied by hydrocephalus that can escape detection by neuroimaging until it is
advanced. It is important to also monitor the opening pressure on lumbar puncture and perform an early shunt to preserve intellectual function. Older patients may develop cord compression from meningeal involvement and benefit from laminectomy.

On chemical analysis of brain tissue, the most striking abnormality is the accumulation of G_M2 ganglioside in cerebral gray matter and cerebellum 100 to 300 times the normal level. The structure of G_M2 ganglioside that accumulates in Tay-Sachs disease is depicted in Fig. 1.19. It consists of ceramide to which 1 mol each of glucose, galactose, N-acetylgalactosamine, and N-acetylneuraminic acid has been attached. Cerebral white matter, liver, spleen, and serum also contain increased amounts of G_M2 ganglioside. The ganglioside content of viscera is not increased. Several other glycosphingolipids are found in lesser amounts.

In most instances, storage of G_M2 ganglioside is the result of a defect in hexosaminidase. Two isozymes of hexosaminidase have been recognized. Hexosaminidases A and B are the two major tissue isozymes; hexosaminidase A is composed of one α subunit and one β subunit, whereas hexosaminidase B is composed of two slightly different β subunits. The gene locus for the α subunit has been mapped to chromosome 15 (15q23–q24); for the β subunit, it is on the long arm of chromosome 5 (5q11.2–q13.3) (595). Both genes have been cloned, and numerous mutations have been described. These are being catalogued on the G_M2 database (http://www.hexdb.mcgill.ca/).

In the classic form of Tay-Sachs disease, the disorder affects the α chain, and hexosaminidase A is inoperative (Fig. 1.19, step A II) in brain, blood, and viscera (596).
Total hexosaminidase activity is usually normal, but the hexosaminidase B component in the CNS acting on its own is unable to hydrolyze G_M2 ganglioside in vivo.

In the Jewish form of generalized G_M2 gangliosidoses three molecular lesions have been found in what had once been considered a pure genetic entity, and a large proportion of patients are compound heterozygotes. The most common mutation, accounting for 73% of Ashkenazi Jews carrying the gene for Tay-Sachs disease in the series of Grebner and Tomczak (597), is a four-base-pair insertion in exon 11 (595). In 18% of cases, there was a single base substitution in intron 12 resulting in defective splicing of the messenger RNA, whereas in 3.3% there was a point mutation on exon 7 (595). In French Canadian patients, an ethnic group in whom the gene frequency for Tay-Sachs disease is equal to that in Ashkenazi Jews, a large deletion in the gene coding for the α chain has been recognized in about 80% of the mutant alleles. The deletion involves part of intron 1, all of exon 1, and probably also the promoter region for the gene. As a consequence, neither mRNA nor immunoprecipitable hexosaminidase A protein is produced. Clinically, this variant is indistinguishable from the Jewish form of Tay-Sachs disease (598).

Mutations in the gene that codes for the gene for the β subunit result in a deficiency of hexosaminidase A and hexosaminidase B, a condition that is termed Sandhoff disease. The most common genetic defect producing this condition is a large deletion in the gene coding for the β subunit (595). Infants with this disorder are non-Jewish, have a mild visceromegaly, and accumulate much larger amounts of globoside (ceramide-glucose-galactose-galactose-galactosamine) in viscera than infants with Tay-Sachs disease (601). Sandhoff disease accounts for approximately 7% of the G_M2 gangliosidoses (600).

A defect at a third gene locus that codes for the G_M2 activator protein produces the AB variant. The action of the activator is to extract a single G_M2 molecule from its micelles to form a water-soluble protein–lipid complex that acts as the true substrate for hexosaminidase A (601). In this entity, accounting for less than 4% of the G_M2 gangliosidoses in the United Kingdom (600), both hexosaminidase components are present but are inactive with respect to hydrolyzing G_M2. The clinical picture of this condition resembles that of classical Tay-Sachs disease. Aside from the G_M2 activator, four other sphingolipid activator proteins (saposins) have been recognized. They are derived from a single precursor protein (prosaposin); their genetically inherited defects lead to other lysosomal storage diseases (602).

Some children who show the clinical picture of late infantile or juvenile amaurotic idiocy (see the section on generalized G_M1 gangliosidosis later in this chapter) have been found to have G_M2 gangliosidosis and a partial defect of hexosaminidase A (603). They account for approximately 25% of English patients with G_M2 gangliosidosis (600). Patients with the adult or chronic form of G_M2 gangliosidosis are compound heterozygotes between one of the infantile mutations and a point mutation at exon 7 in the gene for the α chain (595). The clinical picture is one of a child with learning disability who, over the years, develops a gradually progressive muscular weakness. At times, this picture is complicated by ataxia, at other times by dystonia (604). Finally, there are asymptomatic individuals with little more than 10% residual hexosaminidase A activity (602).

The pathologic changes in Tay-Sachs disease are confined to the nervous system and represent the most fulminant of all the cerebroretinal degenerations. Almost every neuron in the cerebral cortex is distended markedly, its nucleus is displaced to the periphery, and the cytoplasm is filled with lipid-soluble material (Fig. 1.20). A similar substance is stored in the apical dendrites of the pyramidal cells. As the disease progresses, the number of cortical neurons diminishes, with only a few pyknotic cells remaining. The gliotic reaction is often extensive. In the white matter, myelination can be arrested, and in the terminal stages, demyelination, accumulation of lipid breakdown products, and widespread status spongiosus are observed commonly. Similar alterations affect the cerebellar Purkinje cells and, to a somewhat lesser degree, the larger neurons of the brainstem and spinal cord. In the retina, the ganglion cells are distended with lipid. At the margin of the fovea, considerable reduction in the number of ganglion cells and an accumulation of large phagocytic cells occurs.

On electron microscopic examination of the involved neurons, the lipid is found in the membranous cytoplasmic bodies, which are round, oval, and 0.5 to 2.0 μm
in diameter. They occupy a considerable portion of the ganglion cell cytoplasm (Fig. 1.21). The membranous bodies also can be located in axis cylinders, glial cells, and perivascular cells (605). Membranous cytoplasmic bodies consist of aggregates of lipids (90%) and protein (10%). The composition of lipids is approximately one-third to one-half gangliosides (mainly G_M2), approximately 20% phospholipids, and 40% cholesterol. In vitro experiments show membranous cytoplasmic bodies to be formed in neurons as a consequence of high ganglioside concentrations in the presence of phospholipids and cholesterol.

By means of Golgi stains and electron microscopy, massively expanded neuronal processes can be demonstrated even in the earliest stages of Tay-Sachs disease. These meganeurites are found specifically at the axon hillock of the cell and displace distally the initial segment of the axon. With progressive enlargement, they become interspersed between the neuronal soma and the axon. Growth of new neurites is increased, probably as a response to excessive amounts of gangliosides, and dendritic spines are lost (606). Abnormal synapses are formed. As a consequence, GABA-ergic connections are enhanced, and the cholinergic connections are altered. The synaptic alterations can be seen early in the disease, much before ganglioside storage has produced a mechanical disruption of cell cytoplasm and organelles (607). Their presence readily explains the neuronal dysfunction and the early onset of neurologic
deficits. Similar but less prominent meganeurites have been observed in Hurler syndrome, Sanfilippo syndrome, and in some of the other gangliosidoses.

Before the 1970s, when carrier screening became available, the classic form of Tay-Sachs disease was usually confined to Jewish families, particularly those of Eastern European background. Since then the condition has been encountered in a variety of ethnic groups. It is transmitted as an autosomal recessive trait. In the United States, the gene frequency is 1 in 27 among Ashkenazi Jews and 1 in 380 among non-Jews.

Infants appear healthy at birth. Until 3 to 10 months of age, growth and development are essentially unremarkable. Listlessness and irritability are usually the first indications of the illness, as well as hyperacusis in approximately one-half of the infants. Soon thereafter, an arrest in intellectual development and a loss of acquired abilities are observed. Examination at this time shows a generalized hypotonia and what is termed a cherry-red spot in both macular areas. The cherry-red spot at the fovea is due to the red of the choroids being visible through an area of the retina that is relatively free of the white, lipid-swollen ganglion cells. The cherry-red spot is characteristic of Tay-Sachs disease, although it is also seen in the other forms of G_M2 and occasionally is observed in Niemann-Pick disease (types A and C), generalized G_M1 gangliosidosis, Farber lipogranulomatosis, infantile Gaucher disease, metachromatic leukodystrophy, and sialidosis (608). In Tay-Sachs disease, it is invariably present by the time neurologic symptoms have developed.

No significant enlargement of liver or spleen occurs. The neurologic symptoms progress rapidly to complete blindness and loss of all voluntary movements. Pupillary light reflexes remain intact, however. The hypotonia is replaced by spasticity and opisthotonus. Convulsions appear at this time and can be generalized, focal, myoclonic, or gelastic. In the final stages, a progressive enlargement of the head has been observed; it is invariably present if the disease has lasted more than 18 months (609). The condition terminates fatally by the second or third year of life.

The CSF is normal. In the early stages of the disease, neuroimaging shows low density on CT scans in caudate, thalamus and putamen, and cerebral white matter, with increased signal on T2-weighted MRI in these areas (610). The caudate nuclei appear swollen and protrude into the lateral ventricles.

The clinical pictures of Sandhoff disease and the AB form of G_M2 gangliosidosis are similar to that of classic Tay-Sachs disease, except for the presence of organomegaly. Juvenile variants of Sandhoff disease have been described. As a rule, these children have late infantile or juvenile progressive ataxia (611).

Other clinical variants of G_M2 gangliosidosis have been recognized in which the picture is highlighted by motor neuron disease (612) or by a movement disorder (615). As a rule, such patients have traces of hexosaminidase A activity.

The diagnosis of Tay-Sachs disease is made easily on clinical grounds in an infant with a progressive degenerative disease of the nervous system when the infant has the characteristic retinal cherry-red spot and lacks significant visceromegaly. Hexosaminidase assay of serum confirms the clinical impression and also can be used to detect the heterozygote. Prenatal diagnosis by assaying the hexosaminidase A content of amniotic fluid or of fibroblasts cultured from amniotic fluid is possible during the second trimester of gestation (595).

The serum hexosaminidase assay has been automated and used in mass screening surveys. For the Ashkenazi Jewish population, in whom DNA testing identifies 99.9% of carriers, DNA testing is the preferred method for ascertaining carriers (614). For non-Jewish individuals the enzyme assay can be performed initially and, if necessary, followed up by DNA mutation analysis (614). The apparent deficiency of the enzyme in clinically healthy individuals has already been discussed in this section; see Molecular Genetics and Biochemical Pathology.

No effective treatment is known for this condition. The process of G_M2 ganglioside accumulation and subsequent degeneration of brain structure and function is already well established by the second trimester of fetal development, so that postnatal enzyme replacement therapy and bone marrow transplantation cannot be expected to be effective. In fact, trials of bone marrow transplants have only slowed but not halted the progression of Tay-Sachs disease (595). Prevention of lysosomal storage by administration of N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycosphingolipid synthesis, has been found to prevent G_M2 in the brain of mice with Tay-Sachs disease and Sandhoff disease. This approach has been used in the treatment of non-neuropathic Gaucher disease and might be of help in a few patients with some residual hexosaminidase activity (615,616).

Total ganglioside content of brain and viscera is increased, and the stored G_M1 ganglioside constitutes up to 90% of total gray matter gangliosides. Additionally, the asialo derivative of G_M1 (G_A1) is present in large amounts. The ganglioside also is stored in liver and spleen. The ultrastructure of the stored material in the liver, however, is entirely different from the membranous cytoplasmic bodies found in neurons. This difference has been attributed to the accumulation of numerous mannose-containing oligosaccharides, which probably result from defective glycoprotein catabolism (619). It is the storage of these compounds that accounts for the hepatomegaly seen in generalized G_M1 gangliosidosis but not in G_M2 gangliosidosis. A profound deficiency (less than 0.1% of normal) of β-galactosidase, the enzyme that catalyzes the cleavage of the terminal galactose of G_M1 (see Fig. 1.19, step A I), has been demonstrated in several tissues of all patients with G_M1 gangliosidoses regardless of their clinical course (620).

The β-galactosidase is synthesized in a precursor form, which is processed via an intermediate form to the mature enzyme that aggregates in lysosomes as a high-molecular-weight complex of β-galactosidase, a protective protein, and a lysosomal neuraminidase. The β-galactosidase precursor is encoded on the short arm of chromosome 3, and numerous mutations have been recognized, with most patients being compound heterozygotes and the degree of residual β-galactosidase activity determining the clinical course of the disease. In type B Morquio disease, several other mutations of the β-galactosidase gene have been identified (621). It is of note that the same mutation can result in type B Morquio disease and G_M1 gangliosidosis, with the phenotypic expression being dependent on the nature of the second allele with which it is paired (622). Type B Morquio disease is considered in the section on the mucopolysaccharidoses. A combined defect in β-galactosidase and neuraminidase produces a clinical picture of cherry-red spots and myoclonus. It is the consequence of a defective protective protein and considered in the section on sialidase deficiency.

The pathologic picture shows neuronal storage resembling that of G_M2 gangliosidosis (Tay-Sachs disease). The neurons are distended with p-aminosalicylic acid (PAS)–positive lipid material; electron microscopy shows that they contain a large number of membranous cytoplasmic bodies similar to those seen in Tay-Sachs disease (623). Myelin is defective, probably the consequence of oligodendroglial loss and impaired axoplasmic transport (624). Additionally, many unusual membrane-bound organelles appear to be derived from lysosomes. Abnormalities also are seen in the extraneural tissues. In the kidneys, a striking vacuolization of the glomerular epithelial cells and the cells of the proximal convoluted tubules occurs. In the liver, a marked histiocytosis is associated with vacuolization of the parenchymal cells. Visceral storage of G_M1 is less prominent in the slower progressive forms.

For clinical purposes, it is preferable to adhere to the traditional classification of the disease and to divide generalized gangliosidosis into three types. The infantile form of G_M1 is a severe cerebral degenerative disease that can be clinically evident at birth. The infant is hypotonic with a poor sucking reflex and poor psychomotor development. Characteristic facial abnormalities include frontal bossing, depressed nasal bridge, macroglossia, large, low-set ears, and marked hirsutism. Dermal melanocytosis is seen in about one-fourth of patients. This is a condition characterized by persistent or progressive extensive, blue cutaneous pigmentation and indistinct borders in a dorsal and ventral distribution. This skin lesion is characteristic for lysosomal storage disease. The most common lysosomal storage disease associated with dermal melanocytosis is Hurler syndrome (625). Hepatosplenomegaly usually is present after age 6 months. Approximately 50% of patients have cherry-red spots. The skeletal deformities (dysostosis multiplex) are similar to those seen in Hurler syndrome (508,626). Patients with hepatosplenomegaly and rapid neurologic deterioration that begins during the first few months of life, but without facial coarsening or many skeletal deformities, also have been recognized (627).

In the late infantile and juvenile forms of generalized G_M1 gangliosidosis, neurologic symptoms usually become manifest after 1 year of age. Hyperacusis can be striking; seizures, frequently taking the form of myoclonic epilepsy, occur in approximately 50% of the patients, and a slowly progressive mental deterioration develops. Bony abnormalities and hepatosplenomegaly are absent, and optic
atrophy, when present, is usually unaccompanied by a cherry-red spot (628).

In the chronic adult form, progressive intellectual deterioration becomes apparent between 6 and 20 years of age. It is accompanied by ataxia, a variety of involuntary movements, and spasticity. In other cases, the condition is marked by progressive athetosis or dystonia but no dementia (629). Pathologic studies reveal that in this, as well as in other sphingolipidoses, the storage material in the more chronically progressive disorders is localized predominantly to the basal ganglia, notably the caudate and lenticular nuclei (630). In this region, axonal and dendritic changes analogous to those seen in G_M2 gangliosidosis are maximal. The cause for the regional predilection is utterly obscure.

The diagnosis of G_M1 gangliosidosis is suggested by the early onset of clinical manifestations and rapid neurologic deterioration in a patient who has features and radiologic bone changes reminiscent of Hurler syndrome and by the absence of β-galactosidase in conjunctiva, leukocytes, skin, urine, or viscera. Because so many phenotypic variants of this disorder exist, it is advisable to perform a conjunctival biopsy or to assay for β-galactosidase in any patient with an unexplained progressive neurologic disorder. Enzyme assays on fibroblasts cultured from amniotic fluid allow an antenatal diagnosis in offspring of affected families (631).

No treatment is available. G_M1 gangliosidosis has not been treated successfully by bone marrow transplantation or enzyme infusions.

The enzymatic defect in the various forms of Gaucher disease is an inactivity of a lysosomal ceramide glucoside-cleaving enzyme, glycosyl ceramide β-glucosidase (glucocerebrosidase) (635). The gene for this enzyme has been mapped to the long arm of chromosome 1. It has been isolated and sequenced. Numerous mutations have been identified, with two point mutations accounting for some 80% of mutant alleles. As yet, poor correlation exists between the genetic mutation and the ensuing clinical picture, and an individual’s genotype predicts neither the severity nor the course of the illness, although, as a rule, the clinical picture is similar in siblings (636).

As a consequence of the genetic lesion, glucocerebrosidase is defective, and glucocerebroside is the principal sphingolipid stored within the reticuloendothelial system in adult Gaucher disease (637). In affected individuals, the spleen contains greatly increased amounts of ceramide glucoside, ceramide dihexosides, and hematosides (ceramide-glucose-galactose-NANA) (638).

The outstanding feature of all types of Gaucher disease is the widespread presence of large numbers of Gaucher cells in spleen, liver, lymph nodes, and bone marrow (Fig. 1.22). These are modified macrophages, appearing as spherical or oval cells between 20 and 50 μm in diameter with a lacy, striated cytoplasm that contrasts with the vacuolated foam cells of Niemann-Pick disease. Electron microscopy reveals irregular inclusion bodies containing tubular elements (639).

In the neuronopathic form (type 2) of Gaucher disease, the cerebral alterations are of five types. These are foci of acute cell loss with neuronophagia, microglial nodules, and chronic neuronal dropout accompanied by gliosis. Additionally, perivascular Gaucher cells can be seen in white matter, particularly in the subcortical area. They are derived from vascular adventitial histiocytes. Finally, a subtle neuronal cytoplasmic accumulation of a PAS-positive material occurs, which on electron microscopy appears as tubular and fibrillar inclusions, similar to the tubules of isolated glucocerebrosides (640). These are detected principally in the large nerve cells outside the cerebral cortex. A marked degree of cytoplasmic storage is not characteristic of infantile Gaucher disease.

The interrelation between the lipid storage and neuronal cell death is not clear. The selective neuronal loss
in cerebral cortical layers 3 and 5 and hippocampal layers CA2–4 and 4b points to cytotoxic action (641). Lloyd-Evans and coworkers found that elevation of intracellular glucosylceramide results in increased functional calcium stores in cultured neurons. Glucosylceramide as well as glucosylsphingosine and galactosylsphingosine (psychosine), stimulate calcium release from brain microsomes. In the case of glucosylceramide and galactosylsphingosine the calcium release was mediated through the ryanodine receptors (642). This phenomenon may explain some of the neuropathology.

In the neuronopathic (type 2 infantile) form of Gaucher disease, the onset of symptoms is generally noted at age 4 or 5 months with anemia, apathy, and loss of intellectual achievements (643,644). These are followed by a gradually progressive spasticity. Neck retraction and bulbar signs are observed frequently, and the infant can have considerable difficulty in swallowing. An acquired oculomotor apraxia also has been noted in this form as well as in the subacute neuronopathic (type 3) form (645). Splenomegaly is usually quite marked, but liver and lymph nodes might not be enlarged. Pulmonary infiltration owing to aspiration or alveolar consolidation by Gaucher cells can be noted. Occasionally, retinal cherry-red spots are present. Radiographic examination reveals rarefaction at the lower ends of the femora. Laboratory studies show only anemia and thrombocytopenia. A neonatal form of Gaucher disease with complete deficiency of glucocerebrosidase is associated with congenital ichthyosis, hepatosplenomegaly, hydrops fetalis, and a rapidly progressive downhill course. This condition has been noted to have a prenatal onset (646).

The subacute neuronopathic form (type 3) becomes apparent during the first decade of life, manifesting by a slowly progressive hepatosplenomegaly, intellectual deterioration, cerebellar ataxia, and spasticity (647). Myoclonic seizures are common (648). They are accompanied by giant potentials on the somatosensory-evoked potentials (SEP), an indication of abnormal cortical inhibition (649). A horizontal supranuclear gaze palsy can develop early in the disease and was found in almost all patients in the series of Harris and colleagues (645). Neuroimaging studies are generally unremarkable. A disproportionately large number of patients have been encountered in Norrbotten in northern Sweden (650). The condition is caused by a single mutation that has been seen in other parts of the world (651).

The chronic non-neuronopathic (type 1) form of Gaucher disease, which is much more common than the other two forms and which occasionally is clinically evident during infancy, rarely involves the CNS, although some patients can develop an atypical form of parkinsonism and dementia during their adult years (633).

The diagnosis of neuronopathic Gaucher disease must be considered in a child with anemia, splenomegaly, and intellectual deterioration. The presence of Gaucher cells in bone marrow aspirates supports the diagnosis, although Gaucher cells also can be seen in chronic myelocytic leukemia or, occasionally, in thalassemia major. The main difficulty, from a clinical standpoint, is excluding Niemann-Pick disease type A. Assay of glucocerebrosidase in leukocytes and fibroblast cultures, using synthetic or labeled natural substrates, provides a definitive diagnosis and can be used to identify the heterozygote for intrauterine diagnosis of the disease (633).

Two forms of therapy are being used in Gaucher disease: enzyme replacement therapy and substrate reduction therapy. Intravenous infusions of glucocerebrosidase purified from human placenta have been effective in reversing most of the systemic manifestations in the non-neuronopathic and the neuronopathic forms. The neurologic symptoms are less amenable to treatment, and in a series of 21 patients with type 3 disease cognitive function decreased in 8 patients while under therapy, with 3 of these patients developing myoclonic seizures (652). This lack of effectiveness results from the inability of intravenous
administered glucocerebrosidase to cross the blood-brain barrier in significant amounts. Convection-enhanced delivery of the enzyme to the brain has been effective in experimental animals, and could provide a better treatment option (645a). In substrate reduction therapy an inhibitor of glucosylceramide synthase (Miglustat) is used to reduce the rate of substrate formation to a level that can be metabolized by the residual glucocerebrosidase (653).

Bone marrow transplantation has been attempted in the subacute form (type 3) of the disease with an encouraging response in some cases (568). As is the case for the other lysosomal storage diseases, gene therapy using retroviral vectors to transfer the cDNA for the human glucocerebrosidase gene into hematopoietic stem cells is still in the experimental phase.

The basic defect involves α-galactosidase A, an enzyme that is specific for cleavage of the terminal galactose moiety of globotriaosylceramide (ceramide trihexoside), which has an α configuration (see Fig. 1.19, step B II) (659a). The gene for α-galactosidase A is localized to the long arm of the X chromosome (Xq21.33–q22). It has been completely sequenced, and more than 200 mutations have been documented, with almost every family having its own private mutation, making phenotype difficult to predict from genotype (660). Examination of the synthesis and processing of the enzyme indicates that Fabry disease, like most other lysosomal storage diseases, represents a heterogeneous group of mutations affecting enzyme synthesis, processing, and stability. In some patients, deficiency of α-galactosidase A is the result of a complete absence of the protein; in others, the α-galactosidase A polypeptides are synthesized but are rapidly degraded after their transport to lysosomes (661). Yet other patients, with a milder clinical form of the disease, have residual enzyme activity. As a consequence of the enzymatic defect, large amounts of globotriaosylceramide (ceramide-glucose-galactose-galactose), normally present in minute amounts in plasma and kidneys, have been isolated from affected tissues, and lesser quantities of a ceramide dihexoside (ceramide-galactose-galactose) are found in kidney (662).

On pathologic examination, foam cells with vacuolated cytoplasm are found in smooth, striated, and heart musculature; bone marrow; reticuloendothelial system; and renal glomeruli. Much of the pathology of the disorder can be explained by storage within the vascular endothelium.

In the CNS, lipid storage is highly selective and is primarily confined to the lysosomes of vascular endothelium, including that of the choroid plexus. The accumulation of the glycolipid leads to degenerative and proliferative changes and tissue ischemia and infarctions. In some cases, glycolipid also occurs in neurons of the autonomic nervous system, such as the intermediolateral cell columns of the thoracic cord, the dorsal autonomic nuclei of the vagus, hypothalamus, amygdala, and anterior nuclei of the thalamus (663). In these areas, the permeability of the blood–brain barrier can be sufficiently great to allow entrance of the glycolipid (664). On ultrastructural examination, the intraneuronal inclusions resemble those seen in Hurler syndrome, in that zebra bodies are prominent (663).

Fabry disease was first described in 1898 on the basis of its dermatologic lesions (665). It is a rare condition with an incidence of approximately 1 in 40,000 (654). Manifestations usually begin in childhood, but can be delayed into the second or third decade of life. The clinical picture is believed to result from direct involvement of various tissues by lipid deposits or by vascular disease involving the small arteries and arterioles, predominantly in the posterior circulation (666). The disease is a systemic disorder. The first manifestations are usually acroparesthesias, fatigue with exercise, and cold and heat intolerance first appearing in early childhood (657,667). The punctate, angiectatic skin rash, which gave the condition its original name, angiokeratoma corpus diffusum, is commonly the next manifestation, and usually, as in Fabry’s original case, appears in late childhood (668). It is most frequently seen about the hips, umbilicus, and genitalia, but can sometimes assume a butterfly distribution over the face. Diarrhea and postprandial pain are common, and many patients are underweight. Most male patients sweat poorly. The most debilitating symptoms are the acroparesthesial crises in the hands and feet, which become progressively more frequent and can be generalized, lasting for days to weeks, and are accompanied by fever and an elevated sedimentation rate. It is likely that these episodes of acroparesthesia result from peripheral nerve involvement (654). Corneal opacifications, best seen by slit-lamp examination, are common. They have been seen in infancy (669). A peripheral neuropathy affecting the small myelinated and unmyelinated fibers commonly develops as the disease progresses (670). In older patients, hypertension, recurrent cerebral infarction, and hemorrhage are the usual neurologic complications. Neuroimaging studies can be used to delineate the early cerebrovascular alterations consisting of small white matter hyperintense lesions on T2-weighted images and
T1 hyperintensity in the pulvinar (671,672,673). Untreated, the illness is progressive; death is usually caused by renal failure or cardiac dysfunction. Variants atypical through lack of cutaneous manifestations or isolated renal, cardiac, and corneal involvement (cornea verticillata) are fairly common (576).

Heterozygous female patients can be totally asymptomatic or be as severely affected as male patients. Acroparesthesias, exercise intolerance, and cardiac involvement are particularly common (655).

Fabry disease should be considered in children who have intermittent burning pain in their feet, legs, and fingertips, aggravated by warm weather or exercise. In the early stages of the disease, angiomas must be sought with care. The umbilicus and scrotum are the most likely sites (654). In male patients, the diagnosis also can be made by finding lipid-like inclusions in the endothelium and smooth muscle of skin biopsies. Biopsy of the peripheral nerve can reveal swelling and disruption of unmyelinated axons and zebra bodies in perineural fibroblasts (670). The diagnosis in male patients is confirmed by finding a marked deficiency of α-galactosidase A in plasma or serum leukocytes or in cultured skin fibroblasts (670). Female patients must be diagnosed by mutation testing. The disease severity does not correlate with blood enzyme levels in female patients.

Phenytoin, carbamazepine, gabapentin, or a combination of these drugs can be of considerable benefit in relieving the intermittent pain if nonsteroidal antiinflammatory agents are insufficient. Avoidance of extremes of heat and cold, reduction of fevers, and spraying of water on the body (to substitute for sweating) can be helpful measures. Enzyme replacement therapy using 1 mg/kg of α-galactosidase A (Fabrazyme) every 2 weeks reverses systemic manifestations of the disease, stabilizes renal function, and decreases the episodes of acroparesthesia, although improvement in pain may take over a year. These infusions, although very expensive, are well tolerated and are free of major side reactions (674,675,676). In Europe, α-galactosidase B (Replagal), an enzyme functionally identical to α-galactosidase A, is available and infused at the lower dose of 0.2 mg/kg every other week (677). Enzyme replacement therapy improves peripheral nerve function (678) and cardiac function (679) and reduces cerebral hyperperfusion (680,681).

Clinically, NPA is characterized by autosomal recessive transmission with a predilection for Ashkenazi Jewish families [approximately 30% of patients in the series of Crocker and Farber (686)]. Symptoms become apparent during the first year of life with hepatosplenomegaly, which can lead to massive abdominal distention, and with poor physical and mental development. Other systemic symptoms include persistent neonatal jaundice, diarrhea, generalized lymphadenopathy, and pulmonary infiltrates.

In approximately one-third of patients, neurologic symptoms predominate initially, and few children survive
beyond infancy without apparent involvement of the nervous system. Seizures, particularly myoclonic jerks, are common, and marked spasticity can develop before death. Approximately one-half of the infants have hypotonia; with progression of the disease, the nerve conduction velocities slow. On biopsy of the peripheral nerves, the Schwann cells are filled with inclusion bodies (688). Retinal cherry-red spots are found in approximately 25% of patients and can antedate neurologic abnormalities (686). Corneal and lenticular opacifications are common (673a). The progression of the disease is variable, but death usually occurs before age 5 years.

The pathologic hallmark of NPA is the presence of large, lipid-laden cells in the reticuloendothelial system, mostly in spleen, bone marrow, liver, lungs, and lymph nodes. In the brain, massive, generalized deposition of foam cells and ballooned ganglion cells occurs, primarily in the cerebellum, brainstem, and spinal cord (690). Lipid storage also occurs in the endothelium of cerebral blood vessels, in arachnoid cells, and in the connective tissue of the choroid plexus.

Biochemical examination documents the storage of sphingomyelin in affected organs. This compound was first described in 1884 by Thudichum, the father of neurochemistry (691), and was found to have the structure depicted in Fig. 1.23.

Chemical and histochemical studies have shown sphingomyelin to be a major myelin constituent. It is also a normal component of spleen. Sphingomyelinase, which cleaves sphingomyelin into phosphatidylcholine and ceramide, is normally present in liver, kidney, spleen, and brain. Two forms of this enzyme have been distinguished. The lysosomal form has an acidic pH optimum, which distinguishes it from the microsomal form, which has a basic pH optimum. In NPA, the lysosomal enzyme is defective. The gene for lysosomal sphingomyelinase has been mapped to the short arm of chromosome 11p15.1–p15.4. It has been cloned and sequenced, and a variety of mutations have been described. Mutations lacking catalytically active sphingomyelinase result in NPA, whereas mutations that produce a defective enzyme with some residual catalytic activity result in NPB (692). Two common mutations are responsible for more than 50% of Ashkenazi Jewish patients with NPA (693).

Classic NPB is very rare, and the majority of NPB patients develop neurologic symptoms later in life (694). The absence of any childhood neurologic manifestations in NPB precludes its extensive discussion. Suffice it to say that the clinical picture is one of hepatosplenomegaly, hyperlipidemia, and interstitial pulmonary infiltrates. In some cases, sphingomyelin storage occurs in retinal neurons, peripheral nerves, and the endothelium of the cerebral vasculature (695).

Niemann-Pick disease type C (NPC) is more common than NPA and NPB combined and is seen in a variety of ethnic groups. Its prevalence in Western Europe has been estimated at 1 in 150,000, but it is much higher in certain geographic isolates such as in the French Acadians of Nova Scotia. It has been described under several terms, notably Niemann-Pick types E and F, a condition in which vertical supranuclear ophthalmoplegia and sea-blue histiocytes were accompanied by hepatic cirrhosis and juvenile dystonic lipidosis. As can be surmised from the multitude of eponyms, the clinical picture is extremely heterogeneous, even within the same family, and reports describe a continuum of severity, with neurologic symptoms appearing any time from infancy to late adult life. Two genotypes have been recognized. About 95% of patients with NPC have mutations in the NPC1 gene that has been mapped to 18q11; the remainder have mutations in the NPC2 gene mapped to 14q24.3 (696).

The clinical presentation of NPD, now known to be allelic with NPC, is indistinguishable from the slowly progressive form of NPC, except that patients are from southwestern Nova Scotia, a geographic isolate where the heterozygote frequency for this condition ranges between 1 in 4 and 1 in 10 (708,709).

Hepatosplenomegaly, anemia, and failure to thrive in children who show intellectual deterioration can suggest one of the forms of Niemann-Pick disease with neurologic involvement. Leukocytes and skin fibroblasts of patients with NPA are deficient in sphingomyelinase. Sphingomyelinase activity has been found in cultured amniotic fibroblasts, allowing an intrauterine diagnosis of NPA and NPB (695).

Diagnosis of both NPC1 and NPC2 can be made by demonstrating delayed LDL-derived cholesterol esterification and increased amounts of unesterified cholesterol in fibroblasts. Staining with filipin demonstrates the intracellular accumulation of cholesterol (701). A small percentage of patients with NPC show near-normal results of the biochemical tests. Fibroblasts derived from these NPC-variant patients act as in a sphingolipid storage disease and accumulate a fluorescent sphingolipid in their lysosomes rather than in the Golgi complex as is the case in normal cells (710).

The presence of sea-blue histiocytes in the bone marrow also serves to diagnose NPC.

Liver or bone marrow transplantation has been unsuccessful in the treatment of NPA and NPC. Enzyme replacement therapy is being developed for patients with NPB who have no neurologic symptoms. No evidence indicates that dimethylsulfoxide or cholesterol-lowering agents improve neurologic symptoms in NPC.

Although Refsum disease has been known since 1944, when Refsum (772) described two families with polyneuritis, muscular atrophy, ataxia, retinitis pigmentosa, diminution of hearing, and ichthyosis, an underlying disorder in lipid metabolism was uncovered only some 20 years later (773).

The disease usually makes its appearance between ages 4 and 7 years, most commonly with partial, intermittent peripheral neuropathy. This neuropathy can be accompanied by sensorineural deafness, ichthyosis, and cardiomyopathy. The CSF shows albuminocytologic dissociation with a protein level between 100 and 600 mg/dL.

In brain, lipids are deposited in swollen nerve cells and in areas of demyelination with the formation of fatty macrophages (774). The characteristic alterations in peripheral nerve are hypertrophy, sometimes with onion bulb formation, and a loss of myelinated fibers. Electron microscopy reveals Schwann cells to contain paracrystalline inclusions. In all organs, including the brain, quantities of lipids are increased. The lipids contain, as one of their major fatty acids, a branched-chain compound, 3,7,11,15-tetramethylhexadecanoic acid (phytanic acid). Blood levels of phytanic acid are increased. In contrast to normal levels of 0.2 mg/dL or less, they range between 10 and 50 mg/dL. Very long chain fatty acids are normal.

These changes result from a defect in the gene that codes for phytanoyl-coenzyme A hydroxylase. As a consequence, there is a block in the peroxisomal α-oxidation of phytanic to prostanoic acid (Fig. 1.24) (775). Phytanic acid is almost exclusively of exogenous origin and derived mainly from dietary phytol ingested in the form of nuts, spinach, or coffee. When patients are placed on a phytol-free diet, blood phytanic acid levels decrease slowly, and within 1 year reach levels of approximately one-fourth of the original values (776). This change is accompanied by increased nerve conduction velocities, return of reflexes, and improvement in sensation and objective coordination. Periodic plasma exchanges have been used to reduce body
stores of phytanic acid and appear to be particularly effective during the early phases of therapy (776).

The mechanism by which phytanic acid produces the variety of clinical manifestations is not clear. One attractive hypothesis is that its structural similarity to the farnesol and the geranyl-geranyl groups permits phytanic acid to interfere with the formation of cytosolic prenylated proteins and prevents anchoring of cytosolic proteins to membranes.

The differential diagnosis of Refsum disease includes other causes of chronic and intermittent polyneuritis, such as α-lipoprotein deficiency (Tangier disease), which can be diagnosed by examination of the plasma lipoproteins and by the low serum cholesterol levels. Other similar clinical entities are relapsing infectious polyneuritis, the mitochondrial myopathies (ophthalmoplegia plus), acute intermittent porphyria, recurrent exposures to toxins, particularly alcohol or lead, and the various hereditary sensory motor neuropathies described in Chapter 3.

Lafora disease appears between ages 11 and 18 years with the onset of grand mal and myoclonic seizures. At first, the myoclonic seizures are triggered by photic stimulation or proprioceptive impulses and are much more frequent when formal tests of coordination are attempted, simulating the intention tremor of cerebellar ataxia. The EEG is usually abnormal, with generalized and focal and multifocal posterior epileptiform discharges (797). The interval between the bilateral sharp waves and the
myoclonus is 15 ms in the upper extremities and 25 ms in the lower extremities, suggesting that cortical discharges and myoclonic seizures are secondary to a brainstem focus.

With progression of the disease, the major seizures become less frequent, myoclonus increases in intensity, and intellectual deterioration occurs. A terminal stage of dementia, spastic quadriparesis, and almost constant myoclonic seizures is reached within 4 to 10 years of the first symptoms.

The diagnosis of Lafora disease is confirmed by the characteristic polyglucosan storage material in muscle and in the sweat glands (obtained by skin biopsy). In liver, PAS-positive material is found in the extracellular spaces (798). Electron microscopy reveals a disrupted endoplasmic reticulum and large vacuoles containing electron-dense material (799). Storage material can be detected in liver before the appearance of neurologic symptoms (800).

No therapy has been found to arrest the progression of neurologic symptoms. The seizures are generally difficult if not impossible to control with anticonvulsants.

The abnormalities in copper metabolism result in a deposition of the metal in several tissues. In the brain, the largest proportion of copper is located in the subcellular soluble fraction, where it is bound not only to cerebrocuprein, but also to a number of other normal cerebral proteins. Anatomically, the liver shows a focal necrosis that leads to a coarsely nodular, postnecrotic cirrhosis. The nodules vary in size and are separated by bands of fibrous tissues of different widths. Some hepatic cells are enlarged and contain fat droplets, intranuclear glycogen, and clumped pigment granules; other cells are necrotic with regenerative changes in the surrounding parenchyma (885).

Electron microscopic studies indicate that copper is initially spread diffusely within cytoplasm, probably as the monomeric MT complex. Later in the course of the disease, the metal is sequestered within lysosomes, which become increasingly sensitive to rupture. Copper probably initiates and catalyzes oxidation of the lysosomal membrane lipids, resulting in lipofuscin accumulation. Within the kidneys, the tubular epithelial cells can degenerate, and their cytoplasm can contain copper deposits.

In the brain, particularly in patients whose symptoms commenced before the onset of puberty, the basal ganglia show the most striking alterations. They have a brick-red pigmentation; spongy degeneration of the putamen frequently leads to the formation of small cavities (867). Microscopic studies reveal a loss of neurons, axonal degeneration, and large numbers of protoplasmic astrocytes, including giant forms termed Alzheimer cells. These cells are not specific for Wilson disease; they also can be seen in the brains of patients dying in hepatic coma or as a result of argininosuccinic aciduria or other disorders of the ammonia cycle. Opalski cells, also seen in Wilson disease, are generally found in gray matter. They are large cells with a rounded contour and finely granular cytoplasm. They probably represent degenerating astrocytes. In approximately 10% of patients, cortical gray matter and white matter are more affected than the basal ganglia. Here, too, extensive spongy degeneration and proliferation of astrocytes is seen (886). Copper is deposited in the pericapillary area and within astrocytes, but it is uniformly absent from neurons and ground substance.

Lesser degenerative changes are seen in the brainstem, dentate nucleus, substantia nigra, and convolutional white matter. Copper also is found throughout the cornea, particularly the substantia propria, where it is deposited in an alcohol-soluble, and probably chelated, form. In the periphery, the metal appears in granular clumps close to the endothelial surface of Descemet membrane. Here, the deposits are responsible for the appearance of the Kayser-Fleischer ring. The color of the Kayser-Fleischer ring varies from yellow to green to brown. Copper deposition in this area occurs in two or more layers, with particle size and distance between layers influencing the ultimate appearance of the ring (887).

Wilson disease is a progressive condition with a tendency toward temporary clinical improvement and arrest. Its prevalence is about 1 in 30,000 (870). The condition occurs in all races, with a particularly high incidence in Eastern European Jews, in Italians from southern Italy and Sicily, and in people from some of the smaller islands of Japan—groups with a high rate of inbreeding.

In a fair number of cases, primarily in young children, initial symptoms can be hepatic, such as jaundice or portal hypertension, and the disease can assume a rapidly fatal course without any detectable neurologic abnormalities (888,889). In many of these patients, an attack of what appears to be an acute viral hepatitis heralds the onset of the illness (890). The presentation of Wilson disease with hepatic symptoms is common among affected children in the United States. In the series of Werlin and associates, who surveyed patients in the Boston area, the primary mode of presentation was hepatic in 61% of patients younger than age 21 years (891). In approximately 10% of affected children in the United States, Wilson disease presents as an acute or intermittent, Coombs-test-negative, nonspherocytic anemia that is accompanied by leukopenia and thrombocytopenia (891).

When neurologic symptoms predominate, the appearance of the illness is delayed until 10 to 20 years of age, and the disease progresses at a slower rate than in the hepatic form. The youngest reported child with cerebral manifestations of Wilson disease was 4 years old. The first signs are usually bulbar; these can include indistinct speech and difficulty in swallowing. A rapidly progressive dystonic syndrome is not unusual when the disease presents in
childhood. Such patients can present with acute dystonia, rigidity, and fever, with an occasional elevation of serum creatine phosphokinase (892). Rarely, hemiparesis can be the initial presentation (893).

In the experience of Arima and his group, 33% of children presented with hepatic symptoms, mainly jaundice or ascites (888). They were 4 to 12 years old at the time of medical attention. Cerebral symptoms, notably dystonia, drooling, or gait disturbances, were the presenting symptoms in 30% of children. These patients were 9 to 13 years of age. The remainder had a mixed hepatocerebral picture and were 6 to 12 years old at the time of medical attention. Minor intellectual impairment or emotional disturbances also can be observed, but seizures or mental deterioration are not prominent features of the disease.

Before long, the patient has a characteristic appearance. A fixed smile is a result of retraction of the upper lip; the mouth hangs open and drools. Speech is often severely impaired. Tremors are usually quite marked. Though they often are unilateral during the early stages of the disease, sooner or later they become generalized. The tremors are present at rest, but become exaggerated with movements and emotional disturbance. Initially fine, they gradually become coarse as the illness progresses until they assume a characteristic “wing-beating” appearance. Rigidity, contractures, and tonic spasms advance steadily and can involve the extremities. Dementia can be severe in some patients, whereas other patients are merely emotionally labile. A nearly pure Parkinson-like syndrome and progressive choreoathetosis or hemiplegia also have been described. In essence, Wilson disease is a disorder of motor function; despite often widespread cerebral atrophy, no sensory symptoms or reflex alterations occur.

Without treatment, death ensues within 1 to 3 years of the onset of neurologic symptoms and is usually a result of hepatic insufficiency.

The intracorneal, ring-shaped pigmentation first noted by Kayser and Fleischer might be evident to the naked eye or might appear only with slit-lamp examination. The ring can be complete or incomplete and is present in 75% of children who present with hepatic symptoms and in all children who present with cerebral or a combination of cerebral and hepatic symptoms (888). The Kayser-Fleischer ring can antedate overt symptoms of the disease and has been detected even in the presence of normal liver functions. In the large clinical series of Arima, it was never present before 7 years of age (888). “Sunflower” cataracts are less commonly encountered.

CT scans usually reveal ventricular dilation and diffuse atrophy of the cortex, cerebellum, and brainstem. Approximately one-half of the patients have hypodense areas in the thalamus and basal ganglia. Increased density owing to copper deposition is not observed. Generally, MRI correlates better with clinical symptoms than CT. A diagnostic appearance of the MRI has been termed the “face of the giant Panda” (894). This is due to an accentuation of the normal low intensity of the red nucleus and substantia nigra by the surrounding increased intensity of the midbrain and tegmentum. MRI also demonstrates abnormal signals (hypointense on T1-weighted images and hyperintense on T2-weighted images) most commonly in the putamen, thalami, and the head of the caudate nucleus (Fig. 1.30). The midbrain is also abnormal, as are the pons and the cerebellum. Cortical atrophy and focal lesions in cortical white matter also are noted. Correlation with clinical symptoms is not good, in that patients with neurologic symptoms can have normal MRI results and other individuals with no neurologic symptoms can have abnormal MRI results (895,896). Positron emission tomography (PET) demonstrates a widespread depression of glucose metabolism, with the greatest focal hypometabolism in the lenticular nucleus. This abnormality precedes any alteration seen on CT scan (897).

When Wilson disease presents with neurologic manifestations, some of the diagnostic features are the progressive extrapyramidal symptoms commencing after the first decade of life, abnormal liver function, aminoaciduria,
cupriuria, and absent or decreased ceruloplasmin. The presence of a Kayser-Fleischer ring is the most important diagnostic feature; its absence in a child with neurologic symptoms rules out the diagnosis of Wilson disease. The ring is not seen in the majority of presymptomatic patients, nor is it seen in 15% of children in whom Wilson disease presents with hepatic symptoms (890).

An absent or low serum ceruloplasmin level is of lesser diagnostic importance; some 5% to 20% of patients with Wilson disease have normal levels of the copper protein. In affected families, the differential diagnosis between heterozygotes and presymptomatic homozygotes is of utmost importance because it is generally accepted that presymptomatic homozygotes should be treated preventively (898).

The presymptomatic child with Wilson disease can have a Kayser-Fleischer ring (seen in 33% of presymptomatic patients in Walshe series), an increased 24-hour urine copper level (greater than 100 μg/24 hours), hepatosplenomegaly (seen in 38% of cases), and abnormal neuroimaging (seen in approximately one-fourth of cases). Approximately one-half of the patients with presymptomatic Wilson disease had no physical findings, and urinary copper excretion can be normal in children younger than the age of 15 years. Should the diagnosis in a child at risk still be in doubt, an assay of liver copper is indicated (898). Low ceruloplasmin levels in an asymptomatic family member only suggest the presymptomatic stage of the disease; some 10% of heterozygotes have ceruloplasmin levels below 15 mg/dL. Because gene carriers account for approximately 1% of the population, gene carriers with low ceruloplasmin values are seen 40 times more frequently than patients with Wilson disease and low ceruloplasmin values. Therefore, when low ceruloplasmin levels are found on routine screening and are unaccompanied by any abnormality of hepatic function or copper excretion, the individual is most likely a heterozygote for Wilson disease.

When a liver biopsy has been decided on, histologic studies with stains for copper and copper-associated proteins and chemical quantitation for copper are performed. In all confirmed cases of Wilson disease, hepatic copper is greater than 3.9 μmol/g dry weight (237.6 μg/g) as compared with a normal range of 0.2 to 0.6 μmol/g (Fig. 1.31). The finding of normal hepatic copper concentration excludes the diagnosis of Wilson disease (871). Because of the large number of mutations causing the disease, a combination of mutation and linkage analysis is required for prenatal diagnosis. As a rule, this technique is not useful in the diagnosis of an individual patient. However, about one-third of North American Wilson disease patients have a point mutation (His1069Glu), and screening for this mutation can be performed readily (899).

Wilson disease is one of a number of metabolic diseases that can present with extrapyramidal signs and symptoms. These conditions and the best means of diagnosing them are listed in Table 1.26.

All patients with Wilson disease, whether symptomatic or asymptomatic, require treatment. The aims of treatment are initially to remove the toxic amounts of copper and secondarily to prevent tissue reaccumulation of the metal (900,901).

Treatment can be divided into two phases: the initial phase, when toxic copper levels are brought under control, and maintenance therapy. There is no agreed-on regimen for treatment of the new patient with neurologic or psychiatric symptoms. In the past, most centers recommended starting patients on d-penicillamine (600 to 3,000 mg/day). Although this drug is effective in promoting urinary excretion of copper, adverse reactions during the initial and maintenance phases of treatment are seen in approximately 25% of patients. These include worsening of neurologic symptoms during the initial phases of treatment, which frequently is irreversible. Skin rashes, gastrointestinal discomfort, and hair loss are encountered also. During maintenance therapy, one may see polyneuropathy,
polymyositis, and nephropathy. Some of these adverse effects can be prevented by giving pyridoxine (25 mg/day).

Because of these side effects, many institutions now advocate initial therapy with ammonium tetrathiomolybdate (60 to 300 mg/day, administered in six divided doses, three with meals and three between meals). Tetrathiomolybdate forms a complex with protein and copper and when given with food blocks the absorption of copper. The major drawback to using this drug is that it has not been approved for general use in this country.

Triethylene tetramine dihydrochloride (trientine) (250 mg four times a day, given at least 1 hour before or 2 hours after meals) is also a chelator that increases urinary excretion of copper. Its effectiveness is less than that of penicillamine, but the incidence of toxicity and hypersensitivity reactions is lower.

Zinc acetate (50 mg of elemental zinc acetate three times a day) acts by inducing intestinal MT, which has a high affinity for copper and prevents its entrance into blood. Zinc is far less toxic than penicillamine but is much slower acting. Diet does not play an important role in the management of Wilson disease, although Brewer recommends restriction of liver and shellfish during the first year of treatment (901).

Zinc is the optimal drug for maintenance therapy and for the treatment of the presymptomatic patient. Trientine in combination with zinc acetate has been suggested for patients who present in hepatic failure. Liver transplantation can be helpful in the patient who presents in end-stage liver disease. The procedure appears to correct the metabolic defect and can reverse neurologic symptoms (902). Schumacher and colleagues (904a) have also recommended its use for patients with normal liver function, but whose neurological symptoms have not responded to the various chelating agents.

With these regimens, gradual improvement in neurologic symptoms occurs. As a rule, brainstem auditory-evoked potentials improve within 1 month of the onset of therapy, with the somatosensory-evoked responses being somewhat slower to return to normal (903). The Kayser-Fleischer ring begins to fade within 6 to 10 weeks of the onset of therapy and disappears completely in a couple of years (904). Improvement of neurologic symptoms starts 5 to 6 months after therapy has begun and is generally complete in 24 months. As shown by serial neuroimaging studies, a significant regression of lesions occurs within thalamus and basal ganglia (895). Successive biopsies show a reduction in the amount of hepatic copper. Total serum copper and ceruloplasmin levels decrease, and the aminoaciduria and phosphaturia diminish.

As a rule, patients who are started on therapy before the evolution of symptoms remain healthy. Children who have had hepatic disease exclusively do well, and in 80%, hepatic functions return to normal. Approximately 40% of children who present with neurologic symptoms become completely asymptomatic and remain so for 10 or more years. Children with the mixed hepatocerebral picture do poorly. Fewer than 25% recover completely, and approximately 25% continue to deteriorate, often with the appearance of seizures. In all forms of the disease, the earlier the start of therapy, the better is the outlook (888).

When symptom-free patients with Wilson disease discontinue chelation therapy, their hepatic function deteriorates in 9 months to 3 years, a rate that is far more
rapid than deterioration after birth (905). Scheinberg and coworkers postulate that penicillamine not only removes copper from tissue, but also detoxifies the metal by inducing MT synthesis (906).

The characteristic feature of KHD, as expressed in the human infant, is a maldistribution of body copper so that it accumulates to abnormal levels in a form or location that renders it inaccessible for the synthesis of various copper enzymes (909,910). Most of the clinical manifestations can be explained by the low activities of the various copper-containing enzymes. Patients absorb little or no orally administered copper; when the metal is given intravenously, they experience a prompt increase in serum copper and ceruloplasmin (911). Copper levels are low in liver and brain but are elevated in several other tissues, notably intestinal mucosa, muscle, spleen, and kidney. The copper content of cultured fibroblasts, myotubes, or lymphocytes derived from patients with KHD is several times greater than of control cells; however, the kinetics of copper uptake in these cells is normal (912).

ATP7A, the gene for KHD, has been mapped to Xq13.3. It encodes an energy-dependent, copper-transporting P-type membrane ATPase (MNK) (913,914). The ATPase is one of a family of membrane proteins that transports cations across plasma and endoplasmic reticulum membranes. The structural homology between ATP7A and the ATP7B, the gene for Wilson disease, is considerable in the 3′ two-thirds of the genes, but there is much divergence between them in the 5′ one-third (914). ATP7A is expressed in most tissues, including brain, but not in liver. At basal copper levels, the protein (MNK) is located in the trans-Golgi network, the sorting station for proteins exiting from the Golgi apparatus, where it is involved in copper uptake into its lumen (915). At increased intra- and extracellular copper concentrations the MNK protein shifts toward the plasma membrane, presumably to enhance removal of excess copper from the cell (916,917). Numerous mutations have been recognized, and it appears as if almost every family has its own private mutation (918).

As a consequence of the defect in the transport protein, copper becomes inaccessible for the synthesis of ceruloplasmin, superoxide dismutase, and a variety of other copper-containing enzymes, notably ascorbic acid oxidase, cytochrome oxidase, dopamine β-hydroxylase, and lysyl hydroxylase.

Because of the defective activity of these metalloenzymes, a variety of pathologic changes are set into motion. Arteries are tortuous, with irregular lumens and a frayed and split intimal lining (Fig. 1.32A). These abnormalities reflect a failure in elastin and collagen cross-linking caused by dysfunction of the key enzyme for this process, copper-dependent lysyl hydroxylase.

Changes within the brain result from vascular lesions, copper deficiency, or a combination of the two. Extensive focal degeneration of gray matter occurs, with neuronal loss and gliosis and an associated axonal degeneration in white matter. Cellular loss is prominent in the cerebellum. Here, Purkinje cells are hard hit; many are lost, and others show abnormal dendritic arborization (weeping willow) and perisomatic processes. Focal axonal swellings (torpedoes) are observed also (908,919). Electron microscopy often shows a marked increase in the number of mitochondria in the perikaryon of Purkinje cells and to a lesser degree in the neurons of cerebral cortex and the basal ganglia (919). Mitochondria are enlarged, and intramitochondrial electron-dense bodies are present. The pathogenesis of these changes is a matter of controversy.

KHD is a rare disorder; its frequency has been estimated at 1 in 114,000 to 1 in 250,000 live births (920). Baerlocher and Nadal provided a comprehensive review of the clinical features (921). In the classic form of the illness symptoms appear during the neonatal period. Most commonly, one observes hypothermia, poor feeding, and impaired weight gain. Seizures soon become apparent. Marked hypotonia, poor head control, and progressive deterioration of all neurologic functions are seen. The facies has a cherubic appearance with a depressed nasal bridge and reduced movements (922) (Fig. 1.32B). There also is gingival enlargement and delayed eruption of primary teeth. The optic discs are pale, and microcysts of the pigment epithelium are seen (923). The most striking finding is the appearance of the scalp hair; it is colorless and friable. Examination under the microscope reveals a variety of abnormalities, most often pili torti (twisted hair), monilethrix (varying

diameter of hair shafts), and trichorrhexis nodosa (fractures of the hair shaft at regular intervals) (Fig. 1.32C) (923).

Radiography of long bones reveals metaphyseal spurring and a diaphyseal periosteal reaction reminiscent of scurvy (924). The urinary tract is not spared. Hydronephrosis, hydroureter, and bladder diverticula are common (925).

Neuroimaging discloses cerebral atrophy and bilateral ischemic lesions in deep gray matter or in the cortical areas, the consequence of vascular infarctions (926). A progressive tortuosity and enlargement of intracranial vessels also can be shown by MRI angiography. Similar changes are seen in the systemic vasculature (927). Asymptomatic subdural hematomas are almost invariable, and when these occur in conjunction with a skull fracture, the diagnosis of nonaccidental trauma is frequently considered (928,929). EEGs show multifocal paroxysmal discharges or hypsarrhythmia. Visual-evoked potentials are of low amplitude or completely absent (930).

The course is usually inexorably downhill, but the rate of neurologic deterioration varies considerably. There are recurrent infections of the respiratory and urinary tracts, and sepsis and meningitis are fairly common. I have seen a patient in his 20s, and numerous patients have been reported whose clinical manifestations are less severe than those seen in the classic form of KHD, and it appears likely that a continuum in disease severity exists. The correlation between the severity of phenotype and the type of mutation is not good, and variable clinical expressions for identical mutations have been observed (931,932).

One of the most important clinical variants is occipital horn syndrome. As originally described, this condition is characterized by occipital exostoses, which appear as bony horns on each side of the foramen magnum, cutis laxa, and bladder diverticula (933). Mental retardation is frequent but not invariable. The neuropathology of this disease is similar to that seen in the classic forms of KHD (934). Serum copper and ceruloplasmin are usually but not invariably low. A variety of mutations of ATP7A have been described, and in some there is complete absence of the normal gene product (935).

The clinical history and the appearance of the infant should suggest the diagnosis. Serum ceruloplasmin and copper levels are normally low in the neonatal period and do not reach adult levels until 1 month of age. Therefore, these determinations must be performed serially to demonstrate a failure of the expected increase. The diagnosis can best be confirmed by demonstrating the intracellular accumulation of copper and decreased efflux of ⁶⁴Cu from cultured fibroblasts (936). The increased copper content of chorionic villi has been used for first-trimester diagnosis of the disease (936). These analyses require considerable expertise, and only few centers can perform them reliably.

In heterozygotes, areas of pili torti constitute between 30% and 50% of the hair. Less commonly, skin depigmentation is present. Carrier detection by measuring the accumulation of radioactive copper in fibroblasts is possible but is not very reliable (936). The full neurodegenerative disease, accompanied by chromosome X/2 translocation, has been encountered in girls (937).

Trichorrhexis nodosa also can be seen not only in argininosuccinic aciduria and giant axonal neuropathy, but also in a number of other conditions that result in a structural abnormality of the hair shaft. A condition characterized by short stature, ataxia, physical and mental retardation, ichthyotic skin, and brittle hair and nails with reduced content of cysteine-rich matrix proteins has been termed trichothiodystrophy (TTD) (938). Approximately one-half of the patients have photosensitivity. Several genetically distinct entities are included in this group, with DNA repair-deficient TTD resulting from mutations in the DNA repair and transcription factor (939). Other disorders in the hair shaft are reviewed in conjunction with photographs of their microscopic appearance in an article by Whiting (940).

Copper supplementation, using daily injections of copper-histidine, appears to be the most promising treatment. Parenterally administered copper corrects the hepatic copper deficiency and restores serum copper and ceruloplasmin levels to normal. The effectiveness of treatment in arresting or reversing neurologic symptoms probably depends on whether some activity of the copper-transporting enzyme MNK has been preserved and whether copper supplementation has been initiated promptly (936,941). Therefore, it is advisable to commence copper therapy as soon as the diagnosis is established if the child has good neurologic function and to continue therapy until it becomes evident that cerebral degeneration cannot be arrested.

The structure of the gene whose defect is responsible for Lesch-Nyhan syndrome has been elucidated. It codes for the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which is defective in this condition. More than 200 mutations have been recorded; most families studied have their private mutation, and the same mutation is encountered rarely in unrelated individuals (953). Some 85% of these are point mutations or small deletions, and patients produce detectable amounts of normal-sized HGPRT (954). As a consequence of the genetic mutation, HGPRT activity is reduced to less than 0.5% of normal in a number of tissues, including erythrocytes and fibroblast cultures.

Because of HGPRT deficiency, hypoxanthine cannot be reused, and whatever hypoxanthine is formed is either excreted or catabolized to xanthine and uric acid. Additionally, phosphoribosylpyrophosphate, a known regulator of de novo purine synthesis, is increased. For these reasons, de novo uric acid production is increased markedly, and serum urine and CSF uric acid levels are elevated. The excretion of other purines, such as xanthine and hypoxanthine, is increased also (954).

The mechanism by which HGPRT deficiency induces the neurologic disorder is unclear. An abnormality in the dopaminergic system has been well documented. In basal ganglia, notably in the terminal-rich regions of the caudate, putamen, and nucleus accumbens septi, the dopamine concentration is reduced, as are the activities of dopa decarboxylase and tyrosine hydroxylase. Such findings point to a functional loss of a significant proportion of nigrostriatal and mesolimbic dopamine tracts. Using a ligand that binds to dopamine transporters, Wong and coworkers showed a reduction in the density of dopamine-containing neurons (955). A reduction in norepinephrine turnover and a diminution in the function of the striatal cholinergic neurons also have been documented. These alterations of the normal neurotransmitter balance within basal ganglia could account for the movement disorder characteristic of Lesch-Nyhan syndrome (956). Based on animal data, it has been postulated that the self-injurious behavior that is so characteristic of the condition is the consequence of destruction of dopaminergic fibers early in development and subsequent exposure to dopamine agonists (953). Impaired adenosine transport and apoptosis induced by the accumulation of 5′-aminoimidazole-4-carboxamide riboside have also been proposed as contributing to the neurologic manifestations (957,958).

As would be expected from the Lyon hypothesis, the heterozygote female patient has two cell populations, one with full enzymatic activity and the other enzyme deficient. Heterozygotes can be ascertained by determining HGPRT activity in hair follicles.

The morphologic alterations seen in the brain are sparse and can be explained by the uremia, which is often present
terminally (959). The dopamine-producing cells in the substantia nigra appear grossly unaffected.

Clinically, patients with HPRT deficiency fall into three groups. The most severely affected have all the features of classic Lesch-Nyhan disease, another group has neurologic manifestations and hyperuricemia, and yet a third group has isolated hyperuricemia without neurologic deficits (953).

In the classic form of Lesch-Nyhan disease children appear healthy at birth, and initial gross motor milestones are achieved appropriately. During the first year of life, psychomotor retardation becomes evident. Extrapyramidal movements appear between 8 and 24 months of age and persist until obliterated by progressive spasticity. Seizures occur in approximately 50% of the patients. A curious and unexplained feature of the disease is the involuntary self-destructive biting of fingers, arms, and lips, which becomes apparent by 4 years of age. Children are disturbed by their compulsion to self-mutilation and are happier when maintained in restraints. The teeth may have to be removed to prevent damage to the lips and tongue. Hematuria and renal calculi are seen in the majority of individuals, and ultimately renal failure develops. Gouty arthritis and urate tophi are also late complications. A megaloblastic anemia is common. Intellectual levels range from moderate mental retardation to low average (960). In later years, a large proportion of patients develop vocal tics reminiscent of those seen in Tourette disease.

In the second group of patients there is excessive uric acid production, gouty arthritis, and mild neurologic symptoms, most commonly a spinocerebellar syndrome or mild mental retardation, or mild mental retardation, short stature, and spasticity (953,961,962).

The least severely affected group has defective HGPRT with hyperuricemia and renal symptoms but no neurologic deficits (953).

The features of the illness, in particular self-mutilation and extrapyramidal movements, make a diagnosis possible on clinical grounds. Although serum uric acid is usually elevated, diagnosis is best confirmed from the urinary uric acid content, a urinary uric acid to creatine ratio of 3 to 1 or higher being almost diagnostic (953). Enzymatic analyses of lysed erythrocytes, cultured skin fibroblasts, cultured amniotic fluid cells, or other tissue are easily carried out and confirm the diagnosis and can be used for antenatal diagnosis (953,963). Routine MRI studies reveal mild cerebral atrophy. Volumetric MRI shows a one-third reduction in caudate volume (964).

Allopurinol (20 mg/kg per day), a xanthine oxidase inhibitor that blocks the last steps of uric acid synthesis, has been used in treating the renal and arthritic manifestations of the disease. The decrease in uric acid excretion induced by this drug is accompanied by an increase of hypoxanthine and xanthine but does not alter the neurologic manifestations of the disease (953). A variety of drugs such as serotonin agonists or antagonists have been used in an attempt to suppress self-mutilation, none with any clear effect. Chronic stimulation of the globus pallidus has been effective in at least one patient (969). Bone marrow transplantation has been ineffective (953).