Lysosomal free sialic acid storage disorders have a relatively short history. In 1979, three mentally retarded brothers and their female cousin from northeastern Finland were found to suffer from a hitherto unknown lysosomal storage disorder, which was named Salla disease after the birthplace of the patients.12 The clinical presentation included early onset of developmental delay, ataxia, and minor dysmorphic features without signs of visceral or ocular involvement. Laboratory studies revealed slightly increased urinary sialic acid excretion, enlarged lysosomal vacuoles in lymphocytes, and skin biopsy fibroblasts, but normal activities of several lysosomal enzymes. Subsequent studies on the urine of the original four patients and nine other patients with similar clinical presentations revealed a ten- to fifteenfold increase in free sialic acid, but no other abnormal oligosaccharides.13 Studies on cultured fibroblasts indicated that the material stored in the lysosomes was also free sialic acid. A large number of patients, mainly from northeastern Finland, were subsequently detected. Detailed clinical studies enabled the course of the disease to be delineated, from hypotonia in newborns to severe disability in adults with nearly normal life spans.
In 1982, another phenotype was described in severely ill infants with similar, although more pronounced, abnormalities in sialic acid metabolism.14,15 These infants were characterized by failure to thrive, visceral enlargement, edema, and early death. The disease, which was called infantile free sialic acid storage disease, or ISSD, is less frequent than Salla disease and has no ethnic predilection. More than 15 patients with ISSD have been reported in the literature,16,17 in contrast to 95 Salla disease patients in Finland, 27 in Sweden, and a few cases elsewhere. A series of studies since 1986 demonstrate that both diseases are caused by a genetic defect of lysosomal membrane transport of free sialic acid.
Sialic Acid Metabolism in Salla Disease and ISSD
The early recognition that free sialic acid is the storage compound in Salla disease patients prompted metabolic studies of the turnover of this molecule. Using cultured fibroblasts and liver from normal and affected individuals, the major enzymes involved in sialic acid metabolism were shown to be normal. These studies included assays of N-acetylneuraminate lyase, sialidase, CMP-N-acylneuraminate phosphodiesterase, and acylneuraminate cytidylyltransferase.10,18 In fibroblasts, the activity of sialidase was reported to be normal by most investigators, although two authors reported three- to fivefold increases.19,20 Sialidase was also reported as increased in the lymphocytes of one patient with Salla disease.21 All other lysosomal enzymes studied have been normal in ISSD and Salla disease.
The Basic Defect: Impaired Lysosomal Transport of Free Sialic Acid
Several studies in which cultured fibroblasts were loaded with sialic acid demonstrated retention of sialic acid in mutant cells, providing indirect evidence that defective function of the transport system is the primary cause of sialic acid storage disorders. In these experiments, sialic acid precursors,18 low density lipoprotein,22 the methylester of sialic acid,23 fetuin,24 or N-acetylneuraminic acid itself25,26 were used to load the lysosomes with sialic acid. In each case, retention of free sialic acid in the lysosomal fraction could be shown in the cell strains from sialic acid storage disease patients, suggesting a block in the efflux of free sialic acid through the lysosomal membrane. The rate of sialic acid egress was characterized in more detailed studies with isolated lysosomal fractions from fibroblasts preloaded with ManNAc to yield equal sialic acid concentrations in the lysosomes of normal and mutant cell strains.27,28 The initial velocity of egress from normal lysosomes increased linearly with loading. The Q10 for sialic acid transport was 2.4, suggesting a carrier-mediated mode of transport. Patient cell strains displayed a negligible egress velocity regardless of the initial sialic acid loading. These findings, obtained both with Salla disease27 and ISSD cell strains,28 reflect a defective lysosomal membrane transport function in the mutant cells.
Experiments using radiolabeled sialic acid uptake studies in resealed lysosomal membrane vesicles provide direct evidence that a specific carrier facilitates free sialic acid efflux from lysosomes.29 These studies unequivocally prove that a genetic defect of the sialic acid transport system, presumably a protein, is the primary cause of sialic acid storage disorders. Resealed vesicles from purified rat liver lysosomes and impermeable buffers were employed to produce a pH difference across the lysosomal membrane, thereby mimicking in vivo lysosomal conditions. Cotransport of sialic acid with protons produced by an ATP-dependent proton pump provided the physiological mechanism for sialic acid transport. The Km for sialic acid transport under the experimental conditions was approximately 0.2 mM. The sialic acid transport system exhibited the properties of both trans-stimulation and cis-inhibition, demonstrating that it was carrier-mediated. Interestingly, glucuronic acid (GluAc) and some other acidic monosaccharides acted as competitive substrates for the carrier (Table 200-1). Recent studies by the same group further elucidate the characteristics of the transport system and of the putative transport protein itself.30 Using reconstituted proteoliposomes and the radiolabeled glucuronic acid transport assay,31 a small amount of a 57-kDa protein was isolated from rat liver homogenates by a combination of chromatographic techniques. Although the final preparation was 432-fold enriched in transport activity, it represented only a 0.1 percent yield and did not allow amino acid sequencing.
Table 200-1: Substrate Specificity of the Sialic Acid Carrier. |Favorite Table|Download (.pdf) Table 200-1: Substrate Specificity of the Sialic Acid Carrier.
|Inhibitor ||% of Uninhibited Rate |
| Sialic acid analogs || |
|No addition ||100 |
|NeuAc ||10 |
|NeuGc ||12 |
|NeuAc1Me ||49 |
|4MU-NeuAc ||58 |
|NeuAc2en ||50 |
| Other monosaccharides, acidic amino acids || |
|Glucuronate ||10 |
|Gluconate ||14 |
|Galacturonate ||34 |
|Galactonate ||36 |
|Pyruvate ||50 |
|Glucose ||80 |
| N-Acetylglucosamine ||100 |
| N-Acetylmannosamine ||100 |
|Gulonolactone ||88 |
|Mannuronolactone ||86 |
|Aspartate ||89 |
|Glutamate ||86 |
Competitive transport assays using the less-purified preparation revealed that not only acidic monosaccharides, that is, sialic acid, glucuronic acid, and iduronic acid, but also several monocarboxylates, that is, lactate, pyruvate, and valproate, employ the same transport system. The lysosomal monosaccharide transporter appears to share many functional properties with the known monocarboxylate transporters of the plasma membrane, that is, MCT1, MCT2, MCT3, and MEV.32 The carrier for acidic monosaccharides has also been demonstrated in resealed lysosomal vesicles from cultured human fibroblasts and lymphoblasts.33 The transport could be inhibited to the same extent by the addition of either nonradioactive GluAc or sialic acid. Lysosomal vesicle preparations from cell strains of patients with sialic acid storage disorders were almost completely devoid of the transport activity, both with sialic acid and with glucuronic acid. No differences were observed between the transport activities of Salla disease and ISSD cell strains. Further evidence that deficient carrier-mediated transport of sialic acid is the primary genetic defect in these diseases came from studies of obligate heterozygotes. Transport activity in lysosomal preparations from lymphoblast cell strains of Salla disease parents demonstrated intermediate transport rates for glucuronic acid (Fig. 200-3).
Intermediate glucuronic acid transport of obligate carriers of Salla disease. Lysosomal membrane vesicles from controls (C), from patients with Salla disease (P), and from unrelated parents of Salla disease patients (H) were assayed for transport capacity of 0.055 mM [3H] GlcAc (3 μCi) under conditions given in reference33 . (From the Journal of Clinical Investigation, 1991, vol. 87, pp. 1329–35. Used with permission of the American Society for Clinical Investigation.)
The significance of defective lysosomal transport of glucuronic acid to the pathogenesis of sialic acid storage diseases remains unknown. Lysosomal accumulation of GluAC, as well as reduced egress from isolated lysosomal fractions of both Salla disease and ISSD cells, has been reported.34 The amount of glucuronic acid accumulated in the mutant cells averaged only 5 percent of the corresponding amount of free sialic acid. Whether glucuronic acid also accumulates in tissues of patients and is excreted in excess in urine has not been investigated.
A constant finding in both Salla disease and ISSD is the increased urinary excretion (Table 200-2) and cellular concentration of free sialic acid (Table 200-3). Exhaustive characterization of both the urinary excretion product and the storage material in cells almost exclusively identifies N-acetylneuraminic acid.10,13,14,18,19,35 Studies in liver, brain, kidney, placental biopsy, leukocytes, and cultured fibroblasts all give identical results.
Table 200-2: Urinary Free Sialic Acid in Salla Disease, ISSD, and Sialuria Patients |Favorite Table|Download (.pdf) Table 200-2: Urinary Free Sialic Acid in Salla Disease, ISSD, and Sialuria Patients
| ||Free sialic acid (nM/mg creatinine) |
| || |
|Disease ||N ||Mean ± SD ||Range ||Reference |
|Salla ||22 || ||700–2100 ||77 |
| ||39 ||713±406 ||280–1993 ||49 |
|ISSD ||10 ||6002 ||1071–14230 ||15,20,25,25,56,60,78 |
|Sialuria ||4 || ||19000–117000 ||66,69,69a |
|Controls || || || || |
|Adults ||24 || ||7–28 ||13 |
| ||12 ||58±19 ||25–88 ||49 |
|Children ||24 || ||100–650 ||50 |
| ||12 ||131±49 ||71–220 ||49 |
Table 200-3: Free Sialic Acid in Cultured Fibroblasts of Patients with Salla Disease, ISSD, and Sialuria |Favorite Table|Download (.pdf) Table 200-3: Free Sialic Acid in Cultured Fibroblasts of Patients with Salla Disease, ISSD, and Sialuria
| ||Free sialic acid (nM/mg protein) || |
| || |
|Disease ||N ||Mean ± SD ||Range ||Reference |
|Salla ||11 || ||6–33 ||37,77 |
| ||22 ||15.8±10.9 ||4–46 ||49 |
|ISSD ||12 ||75 ||10–269 ||20,37,56,60,78,79 |
|Sialuria ||3 ||143 ||75–273 ||68 |
|Control ||10 ||1.1±1.0 || ||28 |
| ||10 ||2.8±1.8 ||0.3–6.3 ||49 |
In Salla disease, the urinary excretion of free sialic acid is 5 to 20 times normal, and the cellular concentration is 10 to 30 times normal when compared with age-matched controls; in ISSD, there is a twenty- to two-hundredfold increase both in urine and cells (Tables 200-2 and 200-3). In contrast, bound sialic acid levels in the urine and cells of patients have been in the range of controls, or only marginally elevated.
Lysosomal accumulation of free sialic acid has been documented both morphologically and biochemically. Intralysosomal localization of sialic acid was first indicated by immunohistochemical methods using the sialic acid-specific lectin Limulus polyphemus agglutinin in cultured fibroblasts from Salla disease patients.36 Several studies employing cell fractionation of fibroblasts from Salla disease and ISSD patients have demonstrated that the free sialic acid stored in the cells cosediments with the lysosome-enriched fraction.37
In EM studies, typical single membrane-bound vacuoles containing amorphous fibrillogranular material were detected in several organs and in various cell types,12,15,20,21,38,39 including fibrocytes, histiocytes, perithelial cells of capillaries, exocrine and myoepithelial cells of sweat glands, and Schwann cells of peripheral nerves (Fig. 200-4). Lymphocytes and bone marrow cells, as well as hepatocytes and Kupffer cells in hepatic biopsies, have displayed similar storage lysosomes. In two cases of fetuses affected by ISSD, EM studies of chorionic villus biopsies have shown enlarged lysosomal vacuoles supporting the biochemical diagnosis.40,41 Enlarged lysosomes were not present in an 11-week chorionic villus sample obtained for prenatal diagnosis in a family with Salla disease.41
Electron photomicrograph of unmyelinated dermal nerve Schwann cell from a skin biopsy of a patient with Salla disease. Enlarged single membrane-bound vesicles are filled with amorphous fibrillogranular material. × 11,220 (From Archives of Neurology, 1979, vol. 36, pp. 88–94. Used with permission of the American Medical Association.)
Although intralysosomal accumulation of free sialic acid in several organs of patients has been clearly documented, its role in the pathogenesis of the diseases is still largely unknown. Future studies must explain the connection between the severe dysfunction of the brain and free sialic acid storage in the lysosomes.
Genetics and Epidemiology
The mode of inheritance in Salla disease and ISSD is autosomal recessive. Genealogical studies of Salla disease in Finnish families,42 the high rate of consanguineous families with ISSD, the demonstration of half-normal sialic acid transport activity in the lysosomal membrane preparations of obligate heterozygotes for Salla disease,33 and finally the recent molecular studies support this.
The gene coding for the transport protein has recently been cloned and the disease-causing mutations have been identified.43 Earlier, the disease locus was assigned by genetic linkage in Finnish families to chromosome 6q14-15.44 Allelic association and haplotype analysis with closely linked markers further localized the disease gene to a region of approximately 200 kb and gave strong evidence for presence of a founder mutation in the Finnish population.45 This is consistent with the existence of founder mutations in several other autosomal recessive diseases enriched in the Finnish population.46 The common Finnish haplotype found in 94% disease chromosomes was also present in homozygous state in 77% of the Swedish patients with Salla disease, as expected from the known admixture of the two populations. Linkage studies involving ISSD and intermediate phenotype patients having other ethnic backgrounds gave evidence for the same locus on chromosome 6q, suggesting that different phenotypic manifestations of free sialic acid storage are allelic disorders.47
Sialic Acid Transporter Gene.
The chromosomal area harboring the Salla disease gene was further characterized by constructing a physical contig map surrounding the region, thus facilitating the gene identification.48 Using positional cloning strategy and structural homology comparison with known transporter genes, Verheijen and coworkers were recently able to identify a cDNA clone of 2.5 kb which contained an open reading frame of 1485 base pairs.43 The gene was designated the AST gene (for Anion and Sugar Transporter).
The gene is expressed most abundantly in heart, kidney, skeletal muscle, liver, placenta, and brain. The gene product, designated as sialin, is predicted to be 495 amino acids long with twelve transmembrane domains in analogy to many other transporter proteins (Fig. 200-5). Both phenotypes of sialic acid storage were found to harbor mutations in the AST gene. Ninety-five percent of the Finnish Salla disease patients had an R39C missense mutation in both alleles, the rest of them being compound heterozygotes. Parents of Salla disease patients were heterozygotes for the R39C mutation. The ISSD patients so far studied carried a variety of mutations including deletions, insertions, and missense mutations. Altogether 15 mutations of the AST gene have so far been identified.
Putative two-dimensional model of the gene product (sialin) encoded by the AST gene depicting mutations found in Salla disease and ISSD patients. Triangles indicate the six potential N-glycosylation sites. R39C is the prevalent founder mutation of Finnish Salla disease patients. The other mutations are from ISSD patients. (From Nature Genetics, 1999, vol 23, pp 462–465.)
Salla disease displays a strong clustering in the Finnish population, whereas no ethnic predilection exists in the rarer ISSD phenotype. At present, 95 Salla disease patients from 58 Finnish families are known. Most of the families originate from an area in northeastern Finland, but occasionally families come from other areas of the country. An analysis of the birthplace of the grandparents in 45 families with one or more Salla disease patients revealed that 78 of 172 grandparents (45 percent) originated from a region approximately 100 km in diameter in the northeastern part of the country with a present population of approximately 37,000. Several of these families can be traced back to common ancestors.
Twenty-seven patients with the Finnish type of disease are known in Sweden.49 Most of these families are not recent emigrants from Finland; instead, they reflect the admixture of the populations during earlier centuries. It was recently noted that paucity of characteristic clinical abnormalities and lack of appropriate laboratory confirmation may have led to underreporting of Salla disease.50 More than 11 isolated cases in 8 families with the Salla disease phenotype have been described from other countries with a variety of ethnic backgrounds.16 Some of these patients had intermediate phenotypes with clinical findings typical of both Salla disease and ISSD. Since the first description of ISSD in 1982, more than 15 patients have been reported,16 largely from Caucasian families with a wide geographic distribution.
Detailed clinical observations of Salla disease patients have been described for 34 adult42 and 6 pediatric cases.51 Recent clinical and neuroradiologic evaluations from Finland include 41 patients, 19 under age 20 and 22 over age 20 years.49 Additional data, less systematically collected, are available from the rest of the 95 Finnish Salla disease patients. These observations allowed a delineation of the clinical course of the disease (Table 200-4) from the normal-appearing newborn to the severely mentally and physically disabled adult.
Table 200-4: Clinical Findings and Course of the Disease in Patients with Salla Disease |Favorite Table|Download (.pdf) Table 200-4: Clinical Findings and Course of the Disease in Patients with Salla Disease
|Neonatal period || |
|Normal ||6/6 |
|Infancy || |
|Hypotonia ||5/6 |
|Ocular nystagmus ||4/6 |
|Ataxia ||6/6 |
|Sitting at 8 months ||1/6 |
|Walking at 2 years ||1/6 |
|Words at 15 months ||2/6 |
|Childhood || |
|Developmental delay ||34/34 |
|Impaired speech ||34/34 |
|Unable to walk ||8/34 |
|Growth retardation (<-2SD) ||18/34 |
|Adult || |
|Severe mental retardation || |
|IQ 20–40 ||7/34 |
|<20 ||27/34 |
|Ataxia ||34/34 |
|Athetosis ||22/34 |
|Abnormal tendon reflexes ||29/34 |
|Exotrophia ||17/34 |
|Convulsions ||9/34 |
|Age of death (12 cases) ||36 years (range 4–70 years) |
The pregnancy and newborn period are uneventful for Salla disease patients. The first clinical signs of the disease are muscular hypotonia and ataxia, which usually appear at 6 to 9 months of age. Ocular horizontal nystagmus is frequently seen even earlier, and is replaced later by a divergent squint in about half of the cases. One patient exhibited horizontal nystagmus as early as 3 weeks of age.42 The ataxia can be truncal as well as in the extremities. The general health of the infants with Salla disease has been unremarkable, without any increased susceptibility to infections or gastrointestinal disturbances. After the age of 1 year, several patients had brisk tendon reflexes and spasticity in the lower limbs. Motor development is always delayed and approximately one-third of the patients never learn to walk. The mean age at walking among the 41 patients was 4 years (range: 1.2 to 9 years). The ataxia is commonly accompanied by athetosis. Speech development is regularly delayed and impaired, with the age at first words varying from 0.8 to 6.0 years.49 The level of speech development reaches a maximum of that for a 4-year-old child. The majority of patients learned single words or short sentences during the first years of life, but gradual deterioration takes place, and no patient over age 44 could speak even single words. In addition, dysarthria and dyspraxia characterize the speech of all patients; the ability to produce words is affected more than comprehension.
The mental development of Salla disease patients is severely delayed from a young age. By adulthood, patients are severely mentally retarded, with IQs in the range of 20 to 40. The inexorable progression of symptoms that characterizes many lysosomal disorders is not a very prominent feature of Salla disease, except in the late stages of the disease. In fact, the plateau in developmental handicap may obscure the true nature of this progressive disease and delay diagnostic evaluation.
Somatic growth is often retarded in Salla disease patients. Eighteen of 34 cases had a height more than 2 standard deviations below the expected height based on parental height and population averages.42 Two sibs, 21 and 17 years of age, exhibited extreme growth retardation, with heights 8.4 and 5.2 standard deviations below expected. These patients also showed virtually undeveloped secondary sexual features, although most patients have normal pubertal development.
Besides growth retardation and neurologic abnormalities, somatic findings are limited. Facial features (Fig. 200-6) may be coarse in late stages of the disease but to a much lesser extent than in aspartylglucosaminuria and other lysosomal storage disorders. Skeletal dysplasias and other radiologic abnormalities have not been encountered, with the exception of a thickened calvarium present in many patients. Liver and spleen enlargement do not occur despite an abundance of storage lysosomes and excess amounts of free sialic acid in these organs. Corneal clouding and fundal changes, frequent manifestations of lysosomal storage disorders, are absent in Salla disease.
Patients with Salla disease. A, An 18-month-old boy with muscular hypotonia, ataxia, and retarded development, but normal facial features. B, A 28-year-old severely affected female patient.
Neurologic examination can reveal ataxia in the extremities and trunk, often accompanied by athetosis and spasticity. Ataxia is more prominent at younger ages, whereas athetosis and spasticity are more frequent in older patients. Hypotonia is often the leading sign during the first year of life. A constant finding in Salla disease patients over 10 years of age has been a low-voltage-type electroencephalographic recording, which is sometimes seen in younger patients. The severity of clinical symptoms and the age of the patient correlate with the decrease in amplitude. Spike waves have been recorded in patients who have seizures. Generalized convulsions are uncommon in Salla disease, but absence-type episodes of short duration do occur. Sixteen of 41 patients were on antiepileptic treatment. Studies of nerve conduction velocity and somatosensory evoked potentials have given evidence suggesting that the peripheral nervous system is also affected by defective myelination. Half of the patients studied had decreased nerve conduction velocities and three severely affected patients had decreased amplitudes as well.
The life span of patients with Salla disease is relatively long. The oldest patient known to us was 72 years old at the time of his death. The mean age at death for 12 patients has been 34.6 years, but in several cases, the cause of death (e.g., accidents or infections) has been only indirectly related to the disease.
Several neuroradiologic studies have been performed. Early CT scans of the head in Salla disease patients revealed cortical and basal atrophy, more pronounced in old than in young patients. Magnetic resonance imaging (MRI) scans have shed new light on the changes in the central nervous system of Salla disease patients.49,52,53 One girl had a normal brain MRI at 2 months of age, but all other cases have shown abnormalities in myelination. On T2-weighted imaging, a high signal intensity has been observed in white matter areas (Fig. 200-7 A), with myelin present only in the internal capsule, and in patients with a mild clinical presentation, in the posterior periventricular regions as well. Surprisingly, myelination of the cerebellum has been normal. All patients exhibited an abnormally thin, hypoplastic corpus callosum (Fig. 200-7 B). There appears to be a good correlation of severity in phenotype with age of the patient and with MRI abnormalities; severely affected and older patients show cortical and cerebellar atrophy and increased periventricular intensity as a sign of a destructive white matter process.
Myelination defects in Salla disease. A, Axial T2-weighted MR image of a 9-year-old boy with severe Salla disease showing homogeneous, abnormally high signal intensity in the white matter indicating defective myelination. The basal ganglia are normal (1.5T, 2500/90/20). B, Sagittal midline T1-weighted MR image of the same patient illustrating a hypoplastic corpus callosum and atrophic changes, especially in the cerebellum (1.5T, 600/15).
Magnetic resonance spectroscopy (MRS) can yield information about biochemical changes in vivo. MRS was recently performed on 8 Salla disease patients ages 6 to 44 years.49 The N-acetylaspartate signal was consistently increased (34 percent) in the parietal white matter, whereas choline signals were reduced (35 percent) compared with values in healthy individuals. High N-acetylaspartate signals are probably related to the increased sialic acid content of the cells, while low choline signals may indicate defective myelination.
The energy metabolism of the brain was recently investigated using positron emission tomography (PET) scanning with a labeled glucose analog (FDG, 2-fluoro-2-deoxy-D-glucose) as a tracer in nine Salla disease patients presenting with different severities of disease.54 Markedly increased glucose utilization was detected in the frontal and sensorimotor cortices and particularly in the basal ganglia of all patients. On the other hand, cerebellar hypometabolism was seen only in patients with severe ataxia and other clinical presentations of the disease. Reduced energy metabolism thus appears to correlate with severity of the disease, but the increased glucose uptake in the frontal cortex and basal ganglia still requires explanation.
The neuropathology of two patients who died of pneumonia at age 41 was reported.55 The cerebral white matter was severely reduced in both cases, with marked loss of axons and myelin sheaths. Abnormally large amounts of a lipofuscin-like material were seen in the perikaryons of the neurons, particularly in the deep cortical layers. The cerebellum also showed degenerative features and loss of Purkinje cells.
The clinical presentation of ISSD is more fulminant than that of Salla disease and leads to death during the first years of life. The main features and the course of the disease are outlined in Table 200-5 as compiled from the data of the 15 reported cases of ISSD.16,17 Four patients manifested the clinical disease at birth, presenting with ascites, hepatosplenomegaly, and coarse facial features. By the age of 3 months, ISSD patients showed failure to thrive, generalized hypotonia, enlarged liver and spleen, mild bone dysplasias, and coarse facial features with hypopigmented skin and fair hair. Psychomotor development was markedly delayed. The age at death in 10 cases reported in the literature has ranged from 1 month to 5 years (mean: 18.1 months). Autopsy reports of two cases describe severe involvement of the central nervous system.17,56 Widespread neuronal storage was detected with myelin loss, axonal spheroids, and gliosis. Staining with a sialic acid-specific lectin, wheat germ agglutinin, gave direct evidence for sialic acid accumulation in neuronal cells.17 Cardiac muscle cells, renal tubular cells, fat cells, and macrophages from several organs also contained clear vacuoles with electron-lucent material.
Table 200-5: Clinical Findings and Course of the Disease in Patients with ISSD |Favorite Table|Download (.pdf) Table 200-5: Clinical Findings and Course of the Disease in Patients with ISSD
| In Utero || |
|Hydrops fetalis ||2/15 |
|Neonatal period || |
|Ascites and edema ||4/15 |
|Enlarged liver ||8/15 |
|Facial dysmorphism ||8/15 |
|Infancy || |
|Failure to thrive ||15/15 |
|Hypotonia ||8/15 |
|Enlarged liver/spleen ||15/15 |
|Hypopigmentation skin/hair ||10/15 |
|Skeletal changes ||4/15 |
|Growth retardation ||9/9 |
|Age at death (10 cases) || |
|10 months ||(range 1–42 months) |
A unique presentation of ISSD was seen in two Austrian siblings, whose sialic acid storage disease was associated with a steroid-resistant congenital nephrosis.57 On EM of a kidney biopsy, the podocytes, mesangial cells, and endothelial cells displayed numerous membrane-bound vacuoles in addition to typical nephrotic changes. It remains to be seen whether lysosomal sialic acid accumulation can lead to basement membrane dysfunction or whether there exists a specific subtype of ISSD with congenital nephrosis.
A few cases of sialic acid storage disease have been reported with a phenotype intermediate between the classic Salla disease and ISSD.21,35,58 Considerable variation in the severity of the clinical manifestations has also been observed among the Finnish Salla disease patients. The most severely affected patients are characterized by early-onset muscular hypotonia, leading to spasticity in the lower extremities in more advanced stages, severe growth retardation, absent pubertal development, and profound mental retardation. In fact, a phenotypic continuum can be visualized from the classic fetal-onset ISSD, through an early-onset severely disabled and growth-retarded intermediate phenotype, to the classic Salla disease with ataxia and mental retardation but a long life span. Only biochemical and molecular studies of the transport protein or the corresponding gene will reveal whether phenotypically different types represent specific entities (e.g., allelic mutations) or whether other genes and environmental factors contribute to the phenotypic diversity in free sialic acid storage. Haplotype analysis in the Finnish families supports the hypothesis of allelic heterogeneity, because the severe and intermediate phenotypes had haplotypes different from those of the majority of patients with the classic phenotype.52
The diagnosis of both Salla disease and ISSD is based on clinical findings, increased urinary excretion of free sialic acid, and the presence of storage lysosomes in various types of cells and tissues. Recent identification of the transporter gene (AST) and the disease-causing mutations provide possibilities for accurate molecular diagnosis, particularly for Salla disease in the Finnish population with the prevalent founder mutation R39C. The distinct and early clinical findings and the rapid progression in ISSD easily focus the diagnostic studies toward lysosomal disorders. In Salla disease, however, the paucity of clinical findings and the very slow, or even absent, rate of progression of the disease often hampers the specific diagnosis. Several patients, particularly those with no affected sibs, have reached adulthood before diagnostic studies were undertaken. The association of mental retardation, spasticity, and ataxia was often attributed to brain damage caused by an episode of perinatal asphyxia.
Urinary free sialic acid is most commonly demonstrated with thin-layer chromatography.59 Patients constantly excrete excess free sialic acid, which was found in the urine of one patient with Salla disease as early as 3 days of age. The enhanced free sialic acid spot on thin-layer chromatography of a patient's sample is usually distinctive enough for diagnosis (see Chap. 141, Fig. 141-12). Normal newborn infants may also show a pronounced sialic acid spot, warranting further studies. More sophisticated techniques, such as HPLC, need to be applied for quantitative assays of urinary free sialic acid, but for diagnostic purposes, such methods are rarely used.60
ISSD patients excrete approximately 10 times the free sialic acid that Salla disease patients excrete, and the intermediate types fall between the two. There appears to be a correlation between the severity of the clinical manifestations and the level of sialic acid excretion. A similar correlation has been seen between the severity of clinical disease and the amount of sialic acid stored in tissues.
Lysosomal accumulation of free sialic acid can also be demonstrated by finding vacuolated lymphocytes or enlarged lysosomes on EM of a skin or conjunctival biopsy. Vacuolated lymphocytes may not always be present in Salla disease; young patients, in particular, may not have them.51 In ISSD, vacuolated lymphocytes are almost always present.
A skin or conjunctival biopsy serves diagnostic purposes very well because of the variety of cell types present in the specimen. Electron-lucent single membrane-bound lysosomes containing fibrillogranular material with some membrane fragments and occasional dark globules have been seen in fibrocytes, Schwann cells, sweat gland cells, myoepithelial cells, and in capillary epithelial cells (Fig. 200-4). The morphology of the enlarged lysosomes in sialic acid storage disorders is similar to that in other glycoproteinoses, such as mannosidosis and aspartylglucosaminuria, and in various types of mucopolysaccharidoses.
Increased intracellular free sialic acid concentrations in fetal specimens allow prenatal detection of both Salla disease and ISSD using quantitative assays. In addition, the presence of vacuoles on EM study of a first-trimester chorionic villus specimen can support the biochemical diagnosis of ISSD, but probably not of Salla disease. Molecular studies have recently become possible in prenatal studies of sialic acid storage disorders. In all families in which the mutation of the index case (or parental mutations) is known, mutation assay on a chorionic villus specimen provides a reliable means of fetal diagnosis. This is particularly important in Salla disease, in which the increase of free sialic acid in chorionic villus specimens is less pronounced than in ISSD. The high prevalence of the R39C founder mutation in the Finnish population undoubtedly will make the mutation assay the method of choice in prenatal diagnosis of Salla disease. Alternatively, in families in which the disease-causing mutation is unknown, genetic linkage analysis using closely linked markers around the disease locus at 6q14-15 can be applied.
The first prenatal diagnosis of sialic acid storage diseases was reported in 1986 in a family with two previous children affected by ISSD.40 The free sialic acid concentration in cultured amniotic fluid cells, that is, 25.0 nm/mg protein, was approximately 70 times that in control cultures. The cell-free amniotic fluid had a sixfold increase of free sialic acid. The pregnancy was terminated and the diagnosis was confirmed by a nine- to two-hundredfold elevation of free sialic acid in cultured fibroblasts, liver, brain, and kidney of the aborted fetus. Several cell types, including placental trophoblasts, had typical storage lysosomes on EM. At least two other prenatal diagnoses of ISSD have been reported,41,61 and in one of them, the increase of free sialic acid was also shown in an uncultured first-trimester chorionic villus sample.41 The chorionic villus cells had numerous vacuoles on EM.
Salla disease is also amenable to intrauterine diagnosis by assay of free sialic acid in a chorionic villus biopsy or by molecular studies. Cultured amniotic fluid cells are less appropriate for prenatal studies because the elevation of free sialic acid content in those cells can be only moderate, leading to false interpretation. A fivefold increase of free sialic acid was seen in cultured amniotic fluid cells in a pregnancy that led to delivery of a child who was later found to be affected by Salla disease.62 The amniotic fluid supernatant had sialic acid within the range of controls. As in ISSD, sialic acid assay in uncultured first-trimester chorionic villus biopsies has provided reliable prenatal detection of Salla disease.63 Free sialic acid has been 30 to 100 times normal in pregnancies with affected fetuses as compared to pregnancies that had a normal outcome (Table 200-6). Total sialic acid was only slightly elevated, giving a high ratio of free/total sialic acid.
Table 200-6: Prenatal Diagnosis of Salla Disease by Sialic Acid Assay in First Trimester Chorionic Villus Samples (references49 and 63) |Favorite Table|Download (.pdf) Table 200-6: Prenatal Diagnosis of Salla Disease by Sialic Acid Assay in First Trimester Chorionic Villus Samples (references49 and 63)
| ||(nM/mg protein) || || |
| || || || |
| ||Free Sialic Acid ||Total Sialic Acid ||Ratio |
|Outcome || || || |
| ||Mean ||Range ||Mean ||Range ||Mean ||Range |
|Normal (n = 6) ||1.4 ||1.18–2.08 ||15.9 ||14.4–30.0 ||0.07 ||0.05–0.14 |
|Salla (n = 3) ||63.0 ||29.7–115.2 ||84.1 ||54.4–124.8 ||0.69 ||0.55–0.92 |
Heterozygotes for Salla disease and ISSD have no clinical manifestations, and their urinary excretion of free sialic acid is within normal limits. The assay of transport activity with resealed lysosomal vesicles from cultured cells can differentiate between the carriers and normals, but the method is not feasible for clinical purposes.33 The observation of an intermediate free sialic acid level in the granulocyte subpopulation of peripheral blood leukocytes in obligate heterozygotes of Salla disease and ISSD58 requires further confirmation in a larger series of individuals. Mutation assay, if available, provides a reliable carrier test for genetic counseling purposes. A relatively high carrier frequency of the R39C mutation in the Finnish population may justify population-based carrier screening in the future.
Four clinical entities present with intracellular accumulation and urinary excretion of sialic acid.1 Sialidosis is due to deficiency of lysosomal neuraminidase leading to storage of undegraded sialyloligosaccharides (see Chap. 140). Sialidosis patients show evidence of lysosomal accumulation, but the storage material, as well as the urinary excretion product, is bound, not free, sialic acid. Definitive diagnosis of sialidosis can be made by assay of neuraminidase activity in leukocytes or cultured fibroblasts. Galactosialidosis is due to a deficiency of a 32-kDa protective protein, and affected patients also excrete sialyloligosaccharides in urine (see Chap. 140). ISSD and Salla disease are allelic mutations of a gene coding for a lysosomal membrane transport protein, and sialuria is a genetic error of impaired feedback inhibition in the synthesis of sialic acid.
The clinical presentations of infantile sialidosis, galactosialidosis, and ISSD may be very similar during the neonatal period, with hepatosplenomegaly, ascites, edema, and dysmorphic features. Cherry-red spots, which are typical ocular findings in sialidosis and galactosialidosis, are not detectable at this early age. Assay of urinary oligosaccharides and enzyme activities in leukocytes or cultured cells usually leads to the correct diagnosis.
The distinction between ISSD and Salla disease is straightforward in typical cases. The severity of the clinical course of the disease and the difference in the amount of sialic acid excreted in the urine and stored in the tissues usually identify the type of disease. Atypical patients, however, may pose diagnostic difficulties. Visceromegaly, dysmorphic features, and hypopigmentation of the skin point to ISSD because these findings have not been described in Salla disease patients.
The final diagnostic consideration is sialuria, characterized by variable degrees of developmental delay, hepatosplenomegaly, coarse facial features, massive urinary excretion, and cytoplasmic storage of free sialic acid (see “Sialuria,” below).
At present, no specific treatment is available for patients with Salla disease or ISSD. No therapeutic trials with bone marrow transplantation are known. Detailed molecular characterization of the transport mechanism at the lysosomal membrane may pave the way for specific therapeutic modalities.