Am J Hum Genet 31:1-18, 1979
Sialidosis: A Review of Human Neuraminidase Deficiency J. A. LOWDEN' AND J. S. O'BRIEN
INTRODUCTION Defects in neuraminidase activity (N-acetylneuraminic acid hydrolase E. C. 3.2.1.18) have recently been described in several clinical disorders. Cantz et al. [1] and Spranger et al. [2] demonstrated a profound deficiency of acid neuraminidase in a patient who had been previously classified as having mucolipidosis I [3, 4]. The patient had a neurodegenerative disorder with Hurleroid features, skeletal dysplasia, and cherry-red spots in the macula. Signs of the illness developed in the first year of life. In addition to the neuraminidase defect, these patients accumulated sialic acid-containing material in fibroblasts. Parents had activities of neuraminidase which were intermediate between patients and controls, implicating the deficiency as a primary defect. Durand et al. [5], O'Brien [6], Thomas et al. [7], and Rapin et al. [8] have described a group of young adults with neuraminidase deficiency who presented with decreasing visual acuity, cherry-red spots in the macula, myoclonus, and sialyloligosaccharides in the urine. Their phenotype differs from that of mucolipidosis I patients in that intelli- gence is normal, somatic and bony abnormalities are absent, and their survival is longer. Family studies indicate that patients with the "cherry-red spot-myoclonus" syndrome are homozygous for a recessive autosomal mutation affecting acid neuraminidase [6, 7]. An infant with Hurleroid features but normal mental development and acid neuraminidase deficiency has been described by Kelly and Graetz [9]. In addition to these two groups of patients another dysmorphic group of young adults, who were previously thought to have a primary defect in 18-galactosidase activity, has now been included in the clinical spectrum of patients with neuraminidase deficiency [10, 11]. Although these patients are normal at birth and throughout early childhood, dysmorphic features, psychomotor degenerative changes, myoclonus, and frequently a cherry-red spot develop at around 10 years of age. It is timely then to review the clinical phenotype of primary human neuraminidase deficiency. We employ the term "sialidosis" here to describe patients with primary neuraminidase deficiency and believe it should be reserved exclusively for this condition. We also discuss the clinical genetics of sialidosis, the chemistry of the compounds stored, the molecular genetics of acid neuraminidase, the secondary neuraminidase deficiencies in other disorders, the secondary deficiency of f3- galactosidase in some patients with sialidosis, and laboratory methods for diagnosis.
Received July 12, 1978: revised September 10, 1978. This research was supported by grants NS08682 and GM 17702 from the National Institutes of Health. I Both authors: Department of Neurosciences, M-008, University of California, San Diego, La Jolla, California 92093. © 1979 by the American-Society of Human Genetics. 0002-9297/79/3101-0002$01.45 1 2 LOWDEN AND O'BRIEN
CLINICAL PHENOTYPES The 37 patients with proven or probable primary acid neuraminidase deficiency known to us are listed in table 1. To our knowledge table 1 contains no duplications. Two distinct groups are readily identified: (1) sialidosis type 1-a group with normal physical appearance and body proportions, cherry-red spots, myoclonus, and usually normal levels of /-galactosidase in body tissues and fluids; and (2) sialidosis type 2-a dysmorphic group with short stature, bony abnormalities, and often deficient f3- galactosidase in body tissues and fluids. A summary of the pertinent clinical findings in each type is given in table 2. We tabulated all major phenotypic features of the 37 patients in order to subclassify sialidosis type 2 into the infantile and juvenile subgroups; further attempts at subclassification were not productive.
TABLE 1 CONFIRMED OR PROBABLE CASES OF SIALIDOSIS
Authors Year Reference No. Cases Diagnosis Confirmed*
Type 1 -Normosomatic Group:
Wynburn-Mason ...... 1943 [12] 1 ... Symonds ...... 1954 [13] 1 ... Glasgow ...... 1957 [14] 1 ...
... Anderson et al ...... 1958 [15] 2 Tittarellietal ...... 1966 [16] 1 ... Hambert and Peterson ...... 1970 [17] 1 ... Gauzzietal ...... 1973 [18] 2 ... Goldsteinetal ...... 1974 [19] 1 + Justice et al ...... 1976 [20] 1 + Durandetal ...... 1977 [5] 2 +
Rapinetal ...... 1978 [8] 3 + Thomas et al ...... 1978 [7] 2 + Type 2-Dysmorphic Group: Juvenile onset
... Goldbergetal ...... 1971 [21] 3 Oriietai ...... 1972 [22] 1
Loonenetal ...... 1974 [23] 1 +
... Kuriyama et al ...... 1975 [24] 1 Fukunagaetal . 1976 [25] 2 +
... Suzukietal ...... 1977 [26] 4 Okadaetal ...... 1977 [27] 1 + Soggetal ...... 1977 [28] 1 + Infantile onset Sprangeretal ...... 1968 [2] 1 +
Berardetal ...... 1968 [29] 1 + Sprangeretal ...... 1977 [3] 1 + Kelly and Graetz ...... 1977 [9] 1 +
Lowdenetal ...... 1978 [30] 1 +
* Diagnosis was confirmed by enzyme assay for neuraminidase or demonstration of sialyloligosaccharides in urine (see table 3). Probable causes are listed based upon their phenotypic similarity to others in the same category. Recently, G. H. Thomas (American Society of Human Genetics annual meeting, Vancouver, 1978) has demonstrated neuramini- dase deficiency in a patient of Goldberg et al. [21]. SIALIDOSIS: NEURAMINIDASE DEFICIENCY 3 TABLE 2 CLINICAL FEATURES OF SIALIDOSIS
TYPE I TYPE 2 Juvenile Infantile
Age onset ...... 12.4 + 4.7 yrs 10.4 + 5.3 yrs 0- 10 mos Sex ...... 8 Male/10 Female 8 Male/6 Female 2 Male/3 Female Race/ethnic origin ...... 7/10 Italian 10/14 Japanese 5/5 Caucasian Seizures ...... 6/13 5/13 4/5 Myoclonus ...... 16/18 10/13 4/5 Ataxia ...... 3/12 9/12 3/5 Pyramidal tract signs...... 0/18 4/13 3/5 Mental retardation ...... 2/18 8/14 ... Cherry-red spots ...... 17/17 12/12 4/5 Corneal clouding/lens opacity 3/17 10/12 4/5 Loss visual acuity ...... 10/10 11/11 1/1 Hearing loss ...... 0/14 6/10 3/4 Coarse facies ...... 0/14 14/14 5/5 Vertebral changes ...... 0/14 13/13 5/5 Growth disturbance ...... 0/14 12/12 4/5 Visceromegaly ...... 1/14 0/12 5/5 Foam cells ...... 4/8 10/11 5/5 EEG abnormal ...... 6/11 4/7 3/4
NOTE. -All details were not available for all cases, thus the denominator in each figure = no. times this clinical feature was mentioned in case reports.
Sialidosis Type -Normosomatic Group The clinical findings have been well described by Rapin et al. [8]. Patients present at 8-15 yrs with decreasing visual acuity, myoclonus, or both. Examination reveals normal features, normal growth, and usually normal intellect. Cherry-red spots are seen in the macular region of the fundus. The cornea and lens are usually clear, but occasionally punctuate lens opacities are noted [5]. Visual acuity gradually decreases during the course of the disorder. In one patient [8], the cherry-red spots faded in her 20th year. The myoclonus begins in the limbs and is gradually progressive. It is an action-type myoclonus induced by stimulation, movement, or emotion that usually decreases or abates during sleep. As the disease progresses, it may prevent the patient from walking, sitting, and eventually eating. The massive spasms are poorly controlled with anticonvulsants, diazepam, haloperidol, levodopa, conjugated estrogens, ketogenic diet, or magnesium [8, 17]. In association with the progression of the myoclonus, tendon stretch reflexes increase, but as in Fabry disease, several patients complained of burning pain in their extremities which was exacerbated by hot weather [8]. Because of the severity of the myoclonus, the assessment of intellectual capacity is sometimes difficult. Nevertheless, it appears that intelligence is either not impaired or minimally impaired. Grand mal seizures followed by loss of consciousness were seen in six cases but not in seven others. They were often initiated by a massive myoclonic spasm. Electrophysiologic abnormalities were examined extensively in, two patients by Engel et al. [31 ]. They found low voltage fast activity in the EEG. Similar observations were recorded in other cases [8, 17, 19]. 4 LOWDEN AND O'BRIEN One patient had an enlarged liver [5], but all other physical findings were normal in this group. Of tle 10 type 1 patients whose ethnic origins were listed, seven were of Italian ancestry. Two of the eight patients have died; at 21 and 28 years respectively. The oldest living patients are, however, over 30 years [7, 8]. In nine patients, blood smears and/or bone marrow preparations were examined; vacuolated lymphocytes or foam cells were noted in two [5, 19]. Vacuolation was described in the liver of one patient who died with diabetes [18]. Cytoplasmic inclusions and membrane-bound vacuoles were reported in liver biopsies from two patients, one of whom also had foamy histiocytes in the bone marrow [5]. The other had normal bone marrow [8], but a cerebral biopsy revealed cortical thinning and "neuronal lipidosis" with membrano-vesicular and lysosome-like bodies in neurons [32]. Although many cerebral vacuoles appeared empty or slightly filled with amorphous granular material, lipofuscin bodies were also prominent. In frozen sections of liver, the vacuoles stained with Alcian-blue and the periodic-acid Schiff (PAS) reagent, indicating the presence of a negatively charged carbohydrate-containing compound [5, 8]. Many lysosomal hydrolases, including f3-galactosidase, /3-N- acetylhexosaminidase, /3-glucosidase, a-mannosidase, a-fucosidase, /3-glucosidase, ,8-glucuronidase, and sphingomyelinase were assayed in serum, leukocytes, fibro- blasts, and occasionally tissue samples in eight of the patients. In one case ,3- galactosidase activities were less than 10% of normal values in white blood cells and fibroblasts [10], but in all other cases these lysosomal hydrolases were within normal limits. This patient's clinical description is brief [20], but perhaps this child could be better classified as a type 2. Defects in /3-galactosidase activity are common in that group. In eight type 1 patients neuraminidase was assayed, and the enzyme defect demonstrated (table 3). When urines from nine patients were examined for oligosac- charides by thin layer chromatography, the typical increase in resorcinol-positive spots was found (fig. 1). In two cases, oligosaccharides were isolated in quantity from urine, quantified, and structurally characterized [5, 33]. Sialidosis Type 2 -Dysmorphic Group The clinical features in these patients are more variable than in type 1. All present with coarse facial features which vary in severity from thick lips, a flattened nasal bridge, and mild hypertelorism to a typical Hurleroid appearance. The facial changes are often accompanied by increased head circumference and short stature. Radio- graphic changes are similar to those seen in mucopolysaccharidoses but in the juvenile group may be less profound than classic Hurleroid changes; they are usually not seen until other dysmorphic features, such as beaking of the lumbar vertebrae (usually L1 or L2), broadening of the ribs, thickening of the skull, and loss of the fine texture of the bones with increasing coarse trabeculation, become pronounced. Cherry-red spots have been noted in all but one of the cases reviewed. That child was only eight months old when reported [9], and the lesion may not have developed. Myoclonus was present in only 75% of the patients. Ataxia was common in type 2 patients and rare in type 1. SIALIDOSIS: NEURAMINIDASE DEFICIENCY 5 TABLE 3 CONFIRMATORY LABORATORY ASSAYS IN SIALIDOSIS
Author Reference Case No. Sialidase % Control Reference Urinary SOS Reference
Type 1: Rapin et al...... [8] 1 0-19 [34] Elevated [34] 2 0-10 [34] Elevated [34] 3 ...... Elevated [34]
...... Goldstein et al ...... [19] Elevated [34] Durand et al...... [5] 1 10.9 [43] 136 x N [5] 2 13.6 [43] 156 x N [5]
Justice et al...... [20] 1 10 [10] ...... Thomas et al...... [7] 1 3-12 [7] ...... Type 2: Loonen et al...... [23] * 0-2 [11] Elevated [11]
...... Orii et al...... [22] Elevated [22] ...... Okada et al...... [27] Elevated [27] Fukunaga et al...... [25] ...... Elevated L27] Sogg et al...... [28] 0-7 [28] Elevated [28] Spranger et al...... [3] ... 0 ... Elevated [3] Kelly and Graetz ..... [9] ... 2 [9] 6 x N [9] ...... Lowden et al...... [30] Elevated [30]
NoTE.-In many cases laboratory studies on particular patients were not completed at the time of the original case report. References to case reports are cross-indexed to the supporting laboratory data.
Many other Hurleroid features have been observed in the dysmorphic group, including lens opacities, corneal clouding, visceromegaly (particularly liver), joint stiffness, inguinal and umbilical hernias, and sensorineural hearing loss. Most type 2 patients have low normal to moderately retarded intelligence. We have arbitrarily divided the dysmorphic patients into infantile onset and juvenile onset categories. Of the 14 juvenile onset cases, 10 were Japanese patients. The earliest symptoms-joint stiffness and coarse features in some or decreased visual acuity in others-developed between 8-15 years and became progressively more pronounced over the next 10. All but three of the juvenile onset patients are still alive; the oldest is 25. Hepatomegaly was found only in the infantile onset type 2 subgroup. It usually occurs in the absence of clinical splenomegaly and does not result in abnormal liver function tests. Many features of the dysmorphic type of sialidosis are, however, variable and inconsistent (table 2). For example, of the two infants who had dysmorphic signs at birth [9, 30] one grew at the 98th percentile for age and was of apparently normal intelligence, while the other had marked growth and psychomotor retardation, failing to thrive throughout her life. Mental retardation appears to be more common and more severe in type 2 patients. While only two type 1 patients are mildly to moderately retarded [18, 19], five of the juvenile [21-23, 25, 27] and four of the infantile [3, 30] type 2 patients are severely retarded. Various lysosomal hydrolases have been measured in serum, fibroblasts, leukocytes and liver in six juvenile and three infantile cases. In all juvenile cases studied, 6 LOWDEN AND O'BRIEN
FIG. 1.-Thin layer chromatography of oligosaccharides. Urine (10 ILI) or extracts of purified oligosac- charides were spotted on silica gel plates (0.25 mm, Merck, Darmstadt, Silica Gel 60, activated by heating at 1 20TC prior to use) and development was in n-propanol-acetic acid-water 3:2:2 for 2 hr [66]. Plates were sprayed with orcinol (1% in 50%0 H2S04), heated at 120TC for 10 min, and purple staining oligosaccharides were visualized. Normal urine = urine from a normal child; GMI urine = urine from a 2-year-old with GM, gangliosidosis, type 1; gal-octasacch = galactosyloctasaccharide isolated from liver of patient with GM, gangliosidosis; sialidosis urine = urine from patient with sialidosis, type 1; and SOS, Cmpd. A = sialyloligosaccharide containing six sugars (same as Strecker's Cmpd. II, table 5) from urine of sialidosis patient. Monosaccharides migrate to top of chromatogram, and glycoproteins do not migrate in this system. Resorcinol positive sialic acid-containing compounds are outlined in pencil. Note different migration of galactosyl- and sialyloligosaccharides in these disorders.
,3-galactosidase activity was low in tissues but usually normal in plasma. It was normal in two infantile patients [3, 9] and absent in a third [30]. Neuraminidase activities were less than 10% of normal in cultured fibroblasts from two juvenile and three infantile type 2 children (table 3). Excretion of sialyloligosaccharides (SOS) was increased in all urines examined (five juveniles and three infantile) in the type group. Included in the juvenile onset type 2 subgroup was a patient who presented with normal intellect, slightly coarse features, cherry-red spots, myoclonus, and mild bony changes of the osteochondryodystrophy type. Her samples were referred to our laboratory for lysosomal hydrolase assays, and neuraminidase deficiency was documented [28]. Assignation to type 2 was made on the basis of bone changes and short stature (147 cm) at 18 years. The heterogeneous nature of the type 2 group will almost certainly result in further arbitrary classification of other new patients. SIALIDOSIS: NEURAMINIDASE DEFICIENCY 7 Other cases The 37 cases listed in table 1 were uncovered by a literature search for patients with either (1) cherry-red spot-myoclonus syndrome or (2) ,f-galactosidase deficiency with evidence of variability of 13-galactosidase activity in different organs or body fluids. The patient described by Goldberg et al. [21], for example, who was thought to have primary f3-galactosidase deficiency, had normal levels of 8(-galactosidase in liver but only 15% of normal levels in skin biopsies. Furthermore, this patient had bilateral cherry-red spots, which are unusual in either juvenile or adult forms of GM1 gangliosidosis [34-36]. On the other hand, we excluded a case [37] that many authors have discussed in comparison with others in our series. This patient has both cherry-red spots and myoclonus in association with ,8-galactosidase deficiency. Although the total activity is only 3%-5% of the control values in liver and leukocytes, it is 30% of control values in plasma. His parents have 50% of normal /3-galactosidase activity in leukocytes, so he is probably homozygous for a mutation of /8-galactosidase. One patient that we excluded from the type 1 group is a 35-year-old woman who was ill for only 6 weeks [38], presenting with behavioral abnormalities, myoclonus, and terminally a high fever, and loss of consciousness. Although the swollen neurons resembled those in the biopsy of a type 1 patient [36], the history was quite unlike those described above, and the course of her illness was remarkably rapid. In our series we included patients with cherry-red spots but without myoclonus [5] and one patient with neither sign [9]. These patients had both demonstrable sialyloligosacchariduria and neuraminidase deficiency. Undoubtedly, closer examina- tion of patients reported to have adult forms of amaurotic familial idiocy or Niemann-Pick disease will reveal additional cases of sialidosis, and patients with' "mucolipidosis and partial or severe (3-galactosidase deficiency" deserve reexamina- tion. Most of the type 2 patients we list have been so misclassified. We recognize that inclusion or exclusion must depend on the demonstration of the enzyme defect and the presence of SOS in urine or tissues. Only when biochemical diagnosis has been combined with careful clinical description will the full phenotypic spectrum of sialidosis become known.
GENETICS The 18 type 1 patients derive from 13 families. In two families matings were consanguineous. Among the 19 type 2 patients, there are three sets of sibs, and two other patients had affected sibs on whom no details were available. In six families, the parents were consanguineous. Of the 37 patients reviewed almost half (17) were male. To establish the disorder as an autosomal recessive mutation it is customary to demonstrate a gene dose response in obligate heterozygotes. In five families [1, 7, 9, 28, 34] intermediate activity has been demonstrated in parents of probands with sialidosis type 1 (three patients) and type 2 (two patients) [table 4]. Thomas et al. [7] demonstrated intermediate deficiencies of neuraminidase in relatives (niece, nephew, siblings, and children) of probands. These results provide strong evidence that sialidosis is transmitted as an autosomal recessive trait. 8 LOWDEN AND O'BRIEN TABLE 4 NEURAMINIDASE ACTIVITY IN FIBROBLASTS FROM AFFEcTED PATIENTS AND THEIR PARENTS
ENZYME ACTIVITY (nmol/mg cell protein/br) AUTHOR (SIALIDosIs, TYPE) SUBSTRATE Control* Patient Parents
Kelly and Graetz [9] (Type 2, Infantile) ... Fetuin 2.21 (1.04-3.45) .045 1.29, 0.92 MPN 21.7 (11-29) 0 15.2, 23.2 O'Brien [34, 42] (Type 1) ...... NeuNAc 2--6 GalNAc 64 (35-92) 0 20, 29 Soggs et al. [28] (Type 1) ...... MPN 82 7 23, 27 Thomas et al. [7] (Type 1) ...... Fetuin 4.8 (2.2-6.7) 0.6 3.2, 3.2 N-acetyl neuraminlactose (mixed 2-*3 & 2-6) 17 (9.5-24.1) 1.3 1 1.1, 9.0 MPN 25 (12-38) 0.6 8.8, 7.6
Spranger et al. [3] (Type 2, Infantile) ..... Fetuin 2.2 (1.0-3.5) 0.05 1.3, 0.9 MPN 22 (11-29) 0 15, 23
* Values listed are means with range of control values in parentheses.
Seven of 10 patients with type 1 sialidosis are Italian, whereas 10 of the 14 juvenile onset type 2 patients are Japanese. These ethnic predilections suggest increased heterozygote frequencies for this mutation among Italians and Japanese. They also reinforce the validity of the listed groupings. The heterozygote frequencies for type I or 2 sialidosis among Italians or Japanese have yet to be determined. In the infantile onset type 2 disorder, three patients are French, one Portuguese, and in the fifth the background is not listed. Clinically this latter group is the most heterogeneous.
NATURE OF THE STORAGE COMPOUNDS The gamut of compounds stored in sialidosis has not been completely defined. A complete lipid analysis of a brain biopsy of one type 1 patient [32] revealed an increase in cholesterol esters, a slight increase in cerebrosides, and a three- to five-fold increase in the free amino acids in grey matter. The ganglioside content and pattern were normal. In a patient with infantile type 2 sialidosis, lipid analysis of a brain biopsy was normal [29]. In a third patient with juvenile onset type 2 sialidosis [39], lipid analysis of grey matter, including thin layer chromatography of the gangliosides, was normal. Biochemical studies on extraneural tissues, including tissue concentrations of sialyloligosaccharides, have not yet been reported. Durand et al. [5] and Strecker et al. [33] have isolated and characterized 12 oligosaccharides, using urine from patients of Spranger et al. [3], some of which are increased more than 100 times normal. The structures (table 5) are closely related and may derive from a common precursor which could resemble compound X. Significantly, in molar terms, more than 80% of the sialyl linkages are of the a2-*6 configuration. It is believed that these SOS originate as the oligosaccharide side chains of glycoproteins, although only compounds II, III, V, and IX (which contain a2-*6 sialyl linkages) have been demonstrated in glycoproteins [40]. Their appearance in urine as amino acid-free oligosaccharides containing N-acetyl-glucosamine at the reducing end strongly implies the action of an endo-N-acetylglucosaminidase which cleaves the side chain from the glycoprotein at a chitobiose (N-acetylglucosaminyl-N- SIALIDOSIS: NEURAMINIDASE DEFICIENCY 9 W) ~~~~~~0 00 'IC - l 00 - - ) -W- __otCll~~oooo _o oo~_-)6 f o~ ~~ 1Cl - Wk~~~~~~~W 0
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THE NATURE OF THE NEURAMINIDASE DEFECT Neuraminidase deficiency has been demonstrated in more than a dozen patients with sialidosis both in fibroblasts and leukocytes using various substrates (table 6). Comparing assay methods is difficult since conditions vary. Usually assays are performed on whole homogenates prepared by brief sonication of the cells; prolonged sonication causes marked loss of activity [6]. Affected patients have low activity using sialyloligosaccharides (0%-10% of controls) and normal activity using gangliosides. Residual activity is higher in patients' extracts when sialic acid (NeuNAc), linked via an a2-*>3 glycosidic bond to the adjacent hexoside is used as substrate, compared to the a2-*6 linked derivatives. Control fibroblasts also cleave the a2--3 linked derivatives at a higher rate than the a2->6 ones. O'Brien [34] demonstrated a profound deficiency using a-L-N- acetylneuraminosyl (2-*6) N-acetylgalactosaminitol as substrate, indicating that the neuraminidase in question cleaves this linkage. This linkage is common in mucous glycoproteins, and differs from the linkage to galactose, which is frequent in serum glycoproteins. Both O'Brien [6] and Strecker et al. [33, 43] demonstrated that the mutant cells fail to cleave compound II, a naturally excreted sialylhexasaccharide with a a2-*6 linked NeuNAc. Durand et al. [5] and Strecker et al. [33] demonstrated that over 80% of the NeuNAc linkages in the urinary SOS from affected patients are of the a2->6 type and suggested that at least two neuraminidases are present in normal fibroblasts, one hydrolyzing a2->6 linkages and the other hydrolyzing a2->3 linkages, with sialidosis patients having normal activity for the latter. This explanation does not seem tenable for the following reasons. A striking reduction of activity for a2-*3 linked substrates has been demonstrated in sialidosis patients (table 6), indicating that both a2-*3 and a2->6 linkages are cleaved by the same enzyme. Further, parents of sialidosis patients have half normal activity when both pure a2-*3 and a2--6 neuraminlactose are used as substrates [6]. The most tenable explanation is the one gene:one enzyme:many substrates model proposed for /8-galactosidase mutants [44]. The neuraminidase affected by the mutation normally cleaves both a2->3 and a2-*6 linkages, but the mutant enzyme retains higher residual activity for the former, causing more storage and excretion of a2->6 linked oligosaccharides. SIALIDOSIS: NEURAMINIDASE DEFICIENCY 11
TABLE 6 SUBSTRATES USED TO MEASURE NEURAMINIDASE ACrIvITY IN FIBROBLASTS
ENZYME ACTIVITY SUBSTRATE LINKAGE Control (nmoles/mg/hr) Patient (% Control) REFERENCE
MPN ...... 128* 4 [11] 24,29,22 0-19 [6,7,9, 10] NL 2--3 ...... a2--3 89 8.4 [6] NL 2-*6 ...... a2--6 30 3.5 [6] NL Mix (bovine; 85% 2-*3, 15% 2-*6) ...... a2-*3 & a2--*6 24, 19 0-2.8 [3, 9] Fetuin ...... a2-*..3 2.2, 4.5, 8.4 0-10 [3, 7, 9, 10] O'Brien Cmpd. A (same as Strecker Cmpd. II) ...... a2--*6 1.14 0 [6, 43] NeuNAc-GalNAc ...... a2--*6 64 0 [34] GM3 ...... a2--*3 2.3, 3.9, 4.2 100 [10, t] GDla ...... a2--*-3 4.0, 4.9 100 [t]
* Higher activity here is due to different assay conditions from those on next line. t Dr. E. H. Kolodny, personal communication, 1978.
The mutations causing the different types of sialidosis are probably allelic. The different phenotypes may be due to a greater deficiency of neuraminidase in the dysmorphic group (table 4). Predictably this group will excrete and store larger quantities of oligosaccharides (not yet demonstrated). Gangliosides GM3 and GDla (containing only a2-*>3 linked NeuNac) and oligosac- charides are apparently hydrolyzed by different enzymes. No human mutants have been reported with ganglioside neuraminidase deficiency. There are clearly at least two neuraminidase isoenzymes, one active on sialyloligosaccharides, the other on gangliosides. The subcellular distribution and substrate specificity of neuraminidases has been studied extensively. In liver four separate subcellular species of neuraminidase have been described. The enzyme is found in lysosomes [45, 46], plasma membranes [47], Golgi membranes [48], and in the cytosol [49], and all forms hydrolyze a2--3 neuraminlactose. Only the lysosomal and plasma membrane enzymes hydrolyze gangliosides. The pH optima for most neuraminidases is 4.1-4.4, but the soluble enzyme has a pH optimum of 5.5 [49] and appears to be the only form in human serum [50]. Attempts to separate human neuraminidases into different molecular forms have encountered difficulty in solubilizing the enzyme [51]. Tuppy and Palese [52] solubilized pig kidney particulate neuraminidase and found an active band of enzyme migrating on polyacrylamide gels. Venerando et al. [53] separated two active species of neuraminidase from the cytosol of pig brain. These enzymes differed slightly in their pH optima and Km 's. Further studies of the different molecular forms of neuraminidase are necessary to clarify the relationship of each to the defect(s) in sialidosis. 12 LOWDEN AND O'BRIEN EFFECT OF THE NEURAMINIDASE DEFECT ON OTHER LYSOSOMAL HYDROLASES Patients with sialidosis have increased concentrations of bound sialic acid [4, 9]. Some bound neuraminic acid occurs in sialyloligosaccharides (in fibroblasts, from a type 2 patient [11]), but some is also bound to glycoproteins, including lysosomal hydrolases. Using fibroblasts from a type 1 sialidosis patient, D. Swallow (personal communication, 1978), demonstrated that several lysosomal hydrolases and adenosine deaminase have altered electrophoretic mobilities (more electronegative). This charge abnormality is normalized by treatment with neuraminidase. This observation is similar to that of Vladutiu and Rattazzi [54] and Champion and Shows [55] for lysosomal hydrolases in I-cell disease. A major difference however between sialidosis and I-cell disease is that lysosomal hydrolases are not elevated in serum [8] or media [7] in sialidosis as they are in I-cell disease or in mucolipidosis III. Further, treatment of I-cell disease /3-hexosaminidase with neuraminidase corrects only the abnormal charge, not the uptake defect [56] indicating that neuraminidase deficiency is a secondary defect in I-cell disease. The finding that parents of I-cell patients have normal neuraminidase activities supports this proposal [57]. In type 2 sialidosis, a deficiency of acid /8-galactosidase has been described using GM, ganglioside, 4-methylumbelliferyl-,/-D-galactopyranoside, and p-nitrophenyl-,f- D-galactopyranoside as substrates (table 7). This deficiency led to an initial diagnosis of GNll gangliosidosis in at least three sialidosis patients [ 10, 23, 30] and has been used by us to identify many probable cases of sialidosis (table 1). The 83-galactosidase deficiency may be profound in serum, leukocytes and fibroblasts [30] but is often not present in all tissues studied [22]. The mechanism responsible for the /3-galactosidase deficiency is unclear. Enzyme inhibition by sialyloligosaccharides in vitro has not been substantial (J. S. O'Brien,
TABLE 7 COMPARISON OF SIALIDOSIS WITH GM, GANGLIOSIDOSIS AND I-CELL DISEASE
Sialidosis GM I Gangliosidosis I-Cell Disease
CNS Storage ...... Vacuolation MCB's Small vacuoles Granular material Zebra bodies Lipofuscin Lysosomal Hydrolases: Patient .. Sialidase 0-15% Sialidase N Sialidase 0 ,8-Galactosidase N (Type 1) or low (Type 2) f8-Galactosidase 0%- 10% j3-Galactosidase 10%o-30% other Lysosomal hydro- lases low in fibroblasts Lysosomal hydrolases Lysosomal hydrolases in se- in serum rum and media + + + and media N Parents .. Sialidase 50% N Sialidase N Sialidase N ,8-Galactosidase N (3-Galactosidase 50% N 8-Galactosidase N Urine ...... SOS ++++ SOS-O SOS + Galactosyloligosaccharides
NOTE. -N = Normal. SIALIDOSIS: NEURAMINIDASE DEFICIENCY 13 unpublished results). Inactivation is a possibility since /3-galactosidase is irreversibly inactivated by relatively minor manipulations such as heating above 40TC, low concentrations of chloride ions, and slight deviations of pH. These conditions do not affect activity of other lysosomal hydrolases to the same extent. Enzyme leakage does not account for the deficiency since /3-galactosidase is not elevated in serum, media, or urine in sialidosis (J. S. O'Brien, unpublished results). Nonetheless, the molecular basis for diminished /3-galactosidase activity in some patients needs to be explored. One patient with both neuraminidase and ,8-galactosidase deficiency was described as an unusual 83-galactosidase variant and was classified as type IV GM, gangliosidosis [58]. His fibroblasts complemented those from patients with types 1 (and 2) GM1 gangliosidosis in restoring ,8-galactosidase activity, and speculation has arisen regard- ing the molecular basis of the complementation [44, 58]. Immunological studies demonstrated normal quantities of immunoreactive, catalytically inactive 38- galactosidase in his fibroblasts, indicating a structural mutation of /3-galactosidase [59]. Complementation resulting from I-IV fibroblast fusions may be explained by restoration of the type IV /8-galactosidase activity promoted by degradation of NeuNAc-containing compounds by the type 1 neuraminidase whose storage somehow leads to /3-galactosidase inactivation. The same explanation holds for complementation observed by Gravel et a]. (personal communication, 1978), in a patient initially thought to have GM, gangliosidosis [30] but later found to have infantile type 2 sialidosis. Concurrent neuraminidase and /3-galactosidase deficiency was first described by Wenger et al. [10] in a patient with previously diagnosed GM1 gangliosidosis [20]. Failure to find intermediate ,3-galactosidase activities in her parents suggested that the primary defect was not /3-galactosidase deficiency. These authors suggested that the type 4 patient described above may have a secondary 83-galactosidase deficiency. These studies demonstrate the difficulty in distinguishing between a primary defect and a secondary one. They also emphasize that complementation studies must be interpreted with caution. It is not known if the deficiency of 8-galactosidase in type 2 patients is clinically significant. Preliminary evidence suggests it is not since neither GM, ganglioside or galactose-containing oligosaccharides accumulate.
NEURAMINIDASE DEFICIENCY IN OTHER DISORDERS I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III) are closely related disorders in which multiple lysosomal hydrolase deficiencies have been described in fibroblasts, and elevated activities are found in media and serum [60-63]. These changes are not seen in sialidosis. Thomas et al. [57] found no measurable neuraminidase activity in fibroblasts from three patients with I-cell disease; however, their parents had normal fibroblast neuraminidase indicating that the neuraminidase deficiency is not the primary defect. Strecker et al. [43] confirmed the neuraminidase deficiency in leukocytes from two I-cell disease patients but did not study the obligate heterozygotes. Leukocytes from I-cell disease patients usually have low levels (30%-50% of control) of lysosomal hydrolases, but enzyme activity is rarely absent. The enzymes from I-cells appear to 14 LOWDEN AND O'BRIEN lack a recognition factor which is necessary for packaging within lysosomes [64]. Although I-cell- lysosomal enzymes appear to contain increased NeuNAc, the removal of NeuNAc by neuraminidase does not improve uptake, indicating that the neuraminidase deficiency which leads to the charge change of lysosomal hydrolyses is unrelated to the primary defect [56]. The primary defect in I-cell disease may be a deficiency of a Golgi a-mannosidase described by Kress and Miller [65]. This enzyme, which is genetically distinct from lysosomal a-mannosidase, may process lysosomal enzymes after their biosynthesis, cleaving extra mannose residues which must then be removed to uncover the uptake recognition site in the carbohydrate side chains. Failure to remove these mannose residues masks the recognition site for uptake and leads to failure of packaging of several lysosomal hydrolases (including neuraminidase) which bear similar carbohydrate side chains. METHODS FOR DIAGNOSIS A useful screening method for sialidosis is thin layer chromatography of urinary SOS. The method and its application are described by Holmes and O'Brien [66] (fig. 1). Plates are stained for total oligosaccharides with orcinol and specifically for SOS with resorcinol. Differentiation of fucosidosis, mannosidosis, and GM1 gangliosidosis from sialidosis is readily achieved using the appropriate standards. We have now diagnosed eight suspected patients with sialidosis by this method, confirming the diagnosis by enzyme assay. Patients with I-cell disease excrete similar SOS but in quantities which are about 10 times less than in sialidosis [5]. Neuraminidase assays have thus far been carried out using fibroblasts and leuko- cytes. Activity in the latter is approximately 10 times lower than in fibroblasts, but a sensitive assay for leukocytes has been published [67]. The sensitive fluorogenic substrate, 4-methylumbelliferyl-,f-N-acetylneuraminic acid, recently synthesized [68], may be useful for diagnosis and heterozygote detection. Fibroblasts have been the most common tissue source for assay using nonradioactive, commercially available sub- strates. Unfortunately, the best substrates, those with a2--6 linked-NeuNAc [34], are not commercially available but must be prepared. If care is taken to minimize blank values and optimize assay conditions, diagnosis of homozygotes and heterozygotes is relatively straightforward. At present we recommend unnary screening of suspected cases for SOS using a random sample (containing preservative) followed by fibroblast neuraminidase assay of probands and parents to achieve diagnosis. Additional lysosomal enzymes should be assayed to rule out I-cell disease and mucolipidosis III using fibroblasts and serum.
DISCUSSION This is the first review in which an attempt has been made to place clinically dissimilar patients with apparent sialidosis within a single biochemical category. Very likely we have made errors of both omission and inclusion. The increasing use of thin layer chromatography of urinary oligosaccharides and the increasing awareness of neuraminidase mutants in human genetic disease will undoubtedly lead to an expansion of clinical phenotypes. The reports by Horoupian and Ross [69] and Jervis [70], for example, suggest that some cases of late onset amaurotic idiocy may be undiagnosed SIALIDOSIS: NEURAMINIDASE DEFICIENCY 15 cases of sialidosis. The mutant gene frequency of the disorder among Japanese and Italians should be determined. Prenatal diagnosis, not yet explored, is possible. The molecular nature of neuraminidase isoenzymes should be studied. 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