Neutral lipid storage disease (NLSD; also termed multisystem triglyceride storage disorder) is an autosomal recessive disorder characterized by non-membrane-bound cytosolic triacylglycerol droplets in most types of cells that have been examined, including neutrophils (Jordans' anomaly) and those of the liver, muscle, epidermis, dermis, and intestinal mucosa (1,2). Cultured fibroblasts and lymphocytes accumulate triacylglycerol droplets and do not release fatty acids from these triacylglycerol stores. The biochemical defect appears to lie in a recycling pathway that converts stored triacylglycerol to phospholipid (3).
We reviewed the reports of cases in the literature. Vacuolated neutrophils are present in all patients, and ichthyosis was present in 26 of the reported patients, including our patient. In 15 additional patients who lack ichthyosis, the diagnosis is less certain. Clinical manifestations of NLSD include fatty liver, ataxia, mental retardation, neurosensory hearing loss, vacuolated eosinophils, myopathy, cardiomyopathy, and cataracts (1-20). Many patients have been the products of consanguineous unions.
We report a patient with most of the typical features of NLSD and with the additional problem of cholestatic liver disease. Because the ichthyosis in NLSD may be mild or absent, and because the clinical features vary, the diagnosis may be missed in many patients. We have reviewed the reported cases of NLSD and suggest that the diagnosis be considered in patients who have any of the reported features, plus vacuolated neutrophils, which appear to be a universal marker for NLSD.
A healthy girl was born after a full-term pregnancy complicated by three episodes of preterm labor during the final 2 weeks. The parents were brother and sister. Lamellar ichthyosis was noted at birth. She was evaluated at 14 months because of hepatomegaly. Bilateral nuclear cataracts were also noted at that time. Laboratory study results showed elevated activities of serum transaminases, γ-glutamyl transpeptidase, alkaline phosphatase, and lactate dehydrogenase (LDH) (Table 1), with normal levels of electrolytes, lipids, uric acid, bilirubin, ceruloplasmin, and α1-antitrypsin. Serum creatine kinase activity was increased, but strength was normal. Examination of a specimen obtained by needle biopsy of the liver showed macrovesicular fatty change without fibrosis (Fig. 1). Electron micrography showed large non-membrane-bound lipid droplets, normal mitochondria, and normal peroxisomes. Neutrophils and eosinophils, containing large cytosolic vacuoles, were observed on examination of peripheral blood smears (Fig. 2).
During the next 6 years, pruritus persisted. Serum bile acids were 19.9 μM and 10.9 μM on two occasions after she had fasted overnight and were 8.5 μM after a meal (normal, 0-6 μM). The 5′-nucleotidase activity was 16.8 U/l (0.28 μM) (normal, 2-15 U/l). γ-Glutamyl transpeptidase and serum transaminase activities remained elevated, despite treatment with 150 mg ursodeoxycholate twice daily and omega-3 fatty acids (400 mg eicosapentaenoic acid and 300 mg docosahexanoic acid per day). Total bilirubin was never higher than 0.4 mg/dl (7 μmol/l). LDH, alkaline phosphatase, and creatine kinase activities remained variably elevated (Table 1). Although strength remained normal, creatine kinase increased to 2,123 U/l (35 μkat/l) with 2% MB isozyme.
At 5 years, 10 months, physical examination revealed a cooperative child whose height and weight were at the 10th percentile. Her skin was covered with large, plate-like scales, and her scalp was diffusely scaly (Fig. 3). The skin folds, abdomen, and back were hyperpigmented and lichenified. The skin of the palms and soles was thickened. She had moderate pruritus. The hair was normal. The liver edge was firm and rounded and extended 10 cm and 8 cm below the right and left costal margins, respectively. The spleen was not palpable. Motor strength, coordination, and reflexes were normal. She could identify colors and could copy a line and a circle, but not a square or a cross. Speech was clear, and hearing was normal. Formal evaluation showed a developmental delay of approximately 1 year. Laboratory findings included the following normal values: albumin 4.7 g/dl (47 g/l), cholesterol 152 mg/dl (3.93 mM), triglycerides 119 mg/dl (1.3 mM), high-density liporotein cholesterol 44 mg/dl (1.13 mM), and low-density lipoprotein cholesterol 84 mg/dl (2.17 mM).
Cell Culture and Lipid Extraction
Normal human fibroblasts from the American Type Tissue Culture Collection (cell line CCD) and NLSD fibroblasts from the child described above were cultured as described (3). Confluent fibroblast cultures were incubated with 0.5 μCi [14C]-oleic acid plus 0.1 mM sodium oleate in 1% bovine serum albumin (essentially fatty-acid-free) in 10% fetal bovine serum, and with Eagle's minimum essential medium (EMEM) with Earle salts and 1% nonessential amino acids. Cells were then chased in 10% fetal bovine serum, minimum essential medium, and 0.1% bovine serum albumin at 37 °C for 24 hours. At the beginning and end of the chase, the media were removed, the cells were washed, and lipids were extracted (21) and chromatographed on a silica gel G plate in heptaneisopropyl ether-glacial acetic acid (60:40:4 vol/vol/vol). To measure triacylglycerol content, cells were trypsinized from 100-mm dishes, and aliquots of the cellular suspensions were counted. The remaining cells were pelleted by centrifugation, resuspended in water, and probe-sonicated (three pulses of 15 seconds each). Cellular lipids were extracted (21), and the triacylglycerol was determined using a commercial kit (GPO-Trinder, Sigma Co., St. Louis, U.S.A.). DNA was measured fluorometrically (22).
Cultured NLSD fibroblasts contained numerous small lipid droplets that stained with Nile Red (data not shown). The triacylglycerol content of NLSD fibroblasts was 20 times higher than that of normal control fibroblasts (22 nmol/μmol DNA versus 1.13 nmol/μmol DNA), similar to results in previous reports (2,5,23). After 9- and 24-hour chase periods, [14C]-labeled triacylglycerol in normal fibroblasts decreased by 66% and 87%, respectively (Fig. 4). In contrast, NLSD cells did not mobilize any radio-labeled triacylglycerol pool during a 9-hour chase; and at 24 hours, 90% of the initial triacylglycerol remained. Although normal cells recycled some of the labeling from triacylglycerol into phospholipid (15% increase in phospholipids at 9 and 24 hours), the amount of labeled phospholipid in NLSD fibroblasts did not change (Fig. 4). These results are typical of those previously reported for NLSD and indicate a lack of triacylglycerol turnover in NLSD fibroblasts (2,23-25).
REVIEW OF THE LITERATURE
Rozenszajn described the first family with ichthyotic NLSD in 1966 (4). Dorfman reexamined 2 of these patients 8 years later and described an additional 2 unrelated patients (5). The following year Chanarin reported a ninth patient (1), and the disorder has occasionally been termed Dorfman-Chanarin syndrome. Since that time, a total of 26 patients (including the present case) have been reported (Table 2). All 26 had had ichthyosis since birth, and their vacuolated neutrophils contained triacylglycerol. Of these 26 patients representing 18 families, 10 families were consanguineous. Sixty-nine percent of the patients had either elevated serum creatine kinase activity or muscle weakness or both, usually mild and usually first symptomatic in adulthood. Only 2 patients had possible cardiac involvement: An adult had aortic insufficiency, and his son had a heart murmur (9). Although hepatomegaly was described in only 12 patients (46%), fatty liver may be universal, in that an additional 3 patients without enlarged livers had marked steatosis revealed in examination of biopsy specimens. Of the patients with steatosis, 2 had hepatic fibrosis, 1 had cholestasis, and 2 others were said to have pruritus; but no documentation was given. Delayed achievement of milestones, low IQ, or mental retardation was reported in 35%, and small nuclear cataracts were observed in 46%. Neurosensory hearing loss developed, often late, in 38%. Infrequently reported findings include diabetes mellitus (1 patient), ataxia and nystagmus (2 siblings), splenomegaly (2 patients), neurolemmoma (1 patient), and microcephaly (3 patients).
Nonichthyotic NLSD has been described in 18 patients. Vacuolated neutrophils were first described in 1952 (Jordans' anomaly) in two brothers with a rapidly progressive myopathy, cardiomyopathy, and fatty liver (26). The etiology of the brothers' disease was not elucidated. Subsequently, similar features were reported in 8 patients with normal serum and muscle carnitine concentrations and normal muscle carnitine palmitoyltransferase activity (27-30) and in 10 other patients with similar findings but no measurement of carnitine (31-36). The latter patients were thought to have biochemical features of NLSD without ichthyosis (Table 3). Fibroblasts from three of these families retained the [14C]-label in triacylglycerol (27,29), similar to results reported for fibroblasts from NLSD patients with ichthyosis. Thus, biochemically, no difference is apparent in patients who have or lack ichthyosis. Although serum carnitine concentrations were not measured in every nonichthyotic patient, the late onset of skeletal muscle weakness or a cardiomyopathy would seem to rule out a primary defect in carnitine transport. Three of the patients had hepatomegaly, none were retarded, and 2 had neurosensory hearing loss. Although the myopathy in the 3 siblings reported by Snyder (30) improved with carnitine supplementation, the children were not, in fact, carnitine-deficient.
Three infants with NLSD died before the age of 10 months (4,5,7). The life-expectancy of older patients with the disorder is not known, but patients who are 51, 52, and 60 years of age have been reported (Tables 2, 3).
Neutral lipid storage disease is more likely to be diagnosed in patients in childhood if ichthyosis is present. Ichthyotic patients may have a more severe form of NLSD; deafness and mental retardation are also more common in them. In the absence of ichthyosis, NLSD is likely to remain undiagnosed until adulthood when myopathy or cardiomyopathy become clinically apparent.
Biochemically, NLSD appears to be similar in patients with and without ichthyosis. Fibroblasts of NLSD (2,27,23,25), muscle (7), and Epstein-Barr virus (EBV)-transformed lymphocytes (37) have been well characterized biochemically, and all investigated lipid metabolic pathways are unaltered. The uptake, transport, and β-oxidation of fatty acid (2,5,23), three lipase activities (2,24,25), and several glycerolipid synthetic enzyme activities (25) are all normal, as is liver fatty-acid-binding protein (16). Unlike the lysosomal lipase-cholesterol esterase deficiency of Wolman's disease, the defect in NLSD is extralysosomal, and degradation of triacylglycerol synthesized from radio-labeled fatty acid appears to be defective (24,38,39). Although it has been suggested that the cells lack a function required to hydrolyze intracellular triacylglycerol (24,25,39,40), results of recent studies suggest that the defect lies in the ability of NLSD cells to recycle triacylglycerol from lipid droplets to phospholipids (3).
Normal mammalian fibroblasts in culture contain very little triacylglycerol. If the cells are fed excess fatty acids, intracellular triacylglycerol droplets accumulate and are depleted when fatty acids are removed from the media. In contrast, NLSD fibroblasts accumulate triacylglycerol droplets even in the absence of excess media fatty acid. Although one might argue that lack of release of these stored fatty acids might impair cellular energy metabolism, it seems unlikely, as adipose tissue is normal in amount and distribution, and neither mitochondrial nor peroxisomal β-oxidation is impaired. In addition, the clinical symptoms are unlike those of carnitine palmitoyltransferase deficiency II, which appears in adulthood with exercise-induced myoglobinuria and weakness (41). Symptoms also differ from those of carnitine palmitoyltransferase deficiency I, infantile carnitine palmitoyltransferase deficiency II, carnitine transporter deficiency, and medium-chain acyl-CoA dehydrogenase deficiency, all classical blocks of fatty acid β-oxidation that occur in infancy or childhood with severe, episodic, hypoketotic hypoglycemia with fasting, or severe myopathy or cardiomyopathy (41). Hypoglycemia and coma are not features of NLSD, and muscle weakness is generally mild or first occurs in adulthood and is not associated with rhabdomyolysis, myoglobinuria, or cramps, or with exacerbations related to fasting. Finally, the mental retardation present in many NLSD patients is unexplained, because neural tissue does not use fatty acids for energy.
Fatty liver in nonobese children is rare, and may represent injury from toxins, drugs, or genetic diseases. Fatty liver is also seen in patients maintained on parenteral nutrition with excess glucose and can occur in Wilson's disease, cystic fibrosis, and malnutrition. The fatty liver in overweight children is not associated with cholestasis or pruritus (42). Our patient's clinical symptoms were not consistent with any of these diagnoses, and she was neither malnourished nor overweight. Fatty liver is a common response to many genetic defects in energy metabolism, including glycogen storage disease type I (43) and defects in fatty acid oxidation (41). Although the hydrolysis of triacylglycerol in fat cells results from the activation of hormone-sensitive lipase through a cyclic adenosine monophosphate-dependent pathway, it is unclear what regulates the hydrolysis of the triacylglycerol in hepatic lipid droplets. Even less well understood is the relationship these triacylglycerol stores have to β-oxidation, to the synthesis of phospholipids, and to the synthesis of very-low-density lipoproteins (VLDL).
Evidence is emerging that stored triacylglycerol in cells forms a repository of material for phospholipid biosynthesis. For example, in cultured neuroblastoma cells, the glycerol backbone of labeled triacylglycerol moves to newly formed phospholipids (44). Because phosphatidylcholine is required for the biogenesis of (VLDL), choline deficiency in rats results in hepatic steatosis and defective (VLDL) secretion (45). It may be that the macrovesicular fatty deposition in NLSD liver results from a similar lack of phosphatidylcholine for (VLDL) biosynthesis. An alternate possibility is that defective lipid recycling leads to defects in a signal transduction pathway that relies on lipid second messengers, including diacylglycerol, phosphatidic acid, alkylacylglycerols, inositol 1,4,5-triphosphate, platelet activating factor, or the eicosanoids.
The results of clinical phenotype and the biochemical studies in fibroblasts are similar in patients who store triacylglycerol in their cells, regardless of whether they have ichthyosis. Because the clinical manifestations are variable, we suggest that NLSD be considered in patients with unexplained fatty liver or cholestasis, and in those with myopathy or cardiomyopathy in which muscle lipid vacuoles are present. The examiner should look for vacuolated neutrophils that are readily seen on a Wrightstained peripheral smear; these are present in every patient with NLSD. Because vacuolated neutrophils are also observed in some patients with systemic carnitine deficiency (46-48), serum carnitine should be measured in patients who have systemic triacylglycerol storage without ichthyosis. Serum carnitine levels are normal in NLSD.
Acknowledgment: The authors thank Dr. Mary L. Williams for her insightful comments and Dr. Toru Ohue for his help in finding and translating references published in Japanese.
This study was supported by HD19068, HD24570, and a multipurpose Arthritis and Musculoskeletal Diseases Center grant 5-P60 AR3070 from the National Institutes of Health, Bethesda, Maryland.
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