Cholesteryl ester storage disease (CESD) is an autosomal recessive lysosomal storage disorder caused by a variety of mutations of the LIPA gene. These cause reduced activity of lysosomal acid lipase, which results in accumulation of cholesteryl esters in lysosomes. If enzyme activity is very low/absent, presentation is in infancy with failure to thrive, malabsorption, hepatosplenomegaly and rapid early death (Wolman disease). With higher but still low enzyme activity, presentation is later in life with hepatic fibrosis, dyslipidaemia and early atherosclerosis.Identification of this rare disorder is difficult as it is essential to assay leucocyte acid phosphatase activity. An assay using specific inhibitors has now been developed that facilitates measurement in dried blood spots. Treatment of CESD has until now been limited to management of the dyslipidaemia, but this does not influence the liver effects. A new enzyme replacement therapy (Sebelipase) has now been developed that could change treatment options for the future.
- liver disease
- inherited pathology
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Cholesteryl ester storage disease (CESD) is a rare lysosomal storage disease that leads to accelerated atherosclerosis and liver disease, including cirrhosis. It is a very rare condition, which consequently may be overlooked as a potential diagnosis. While in the past, this would have a minimal effect for patients because there was no definitive treatment, although control of serum lipids helps, a new treatment is now being developed, and so, identification of individuals with the condition is important to avoid preventable morbidity and mortality. This paper describes the disease biological, epidemiological, genetic, pathological and radiological features, screening and treatment options.
Wolman disease (WD) and CESD represent two distinct phenotypes caused by the same basic defect: a complete or partial deficiency of lysosomal acid lipase (LAL), which is encoded by the LIPA gene.1 WD is characterised by generalised lipid storage with hepatosplenomegaly, abdominal distension, ascites, intestinal malabsorption with steatorrhoea and failure to thrive; it is rapidly progressive, lethal and presents in early infancy.2 ,3 CESD is characterised by hepatic fibrosis, hyperlipidaemia and accelerated atherosclerosis and may be diagnosed at any age from a few years old. There are no specific features that could be used to distinguish CESD from non-alcoholic fatty liver disease.
It appears that the two variants are the result of a variation in the residual LAL activity.4 Structural modelling of the proteins resulting from various mutations indicates that residues responsible for WD/CESD have reduced solvent accessibility and that this results in protein conformation changes with more severe changes being associated with WD and lesser changes with CESD.5
The function of LAL is to hydrolyse cholesteryl esters in lysosomes, although as with most enzymes under certain conditions it is possible that the reverse function may catalyse esterification. Deficiency in LAL therefore results in a failure of hydrolysis and build-up of undigested triglycerides and cholesteryl esters in the cell. When LAL activity is below 5% of normal, the rate of release of cholesterol from late endosomes/lysosomes falls below a critical level and intracellular regulation of ATP-binding cassette transporter A1 (ABCA1), which is primarily controlled by oxysterol-dependent activation of liver X receptors (LXR), fails. This causes decreased loading of phospholipid and cholesterol to apolipoprotein A-1, reduced α-high-density lipoprotein (HDL) particle formation and consequent hypoalphalipoproteinaemia6 (figure 1).
In Niemann–Pick disease type C1, sphingomyelinase deficiency results in accumulation of sphingolipids in lysosomes with disruption of LXR and consequent reduced HDL due to changes in expression of ABCA1.6
It is also hypothesised that oxidation of low-density lipoprotein (LDL) particles in patients with WD is a critical part of the disease mechanism. In cultured WD adrenal cells, mildly oxidised LDL is absorbed via LDL receptors and cannot be metabolised in lysosomes. This causes a sustained rise in intracellular Ca++ ions and subsequent cell death with deposition of calcium.7 Calcification of the adrenal glands is a significant feature of WD.
A mouse model of LAL deficiency has been created.8 Homozygous lal-/-mice appear normal at birth but there is massive triglyceride and cholesteryl ester storage in adult liver, adrenal and small intestine. Death tends to occur at around 7–9 months of age. At 6–8 months of age, the white adipose tissue is entirely absent and there is progressive loss of brown adipose tissue. Clinical features of lipid storage begin to appear early in these animals, with the liver being 1.5−2× its normal size and of a yellowish hue by 21 days of age.9 Lipid accumulation is approximately 30× normal.
One difficulty with establishing the frequency of CESD/WD is that because it is rare, it is rarely considered as a potential diagnosis. Consequently, it is quite possible that many affected individuals are incorrectly diagnosed with non-alcoholic steatohepatitis.
According to the literature, approximately 50% of cases of CESD reported have the Exon 8 splice junction mutation (E8SJM).10 By assessing the prevalence of that mutation in a test cohort of 1152 individuals from the PROCAM-Study of North-Western Germany and two validation cohorts, Muntoni et al estimate that according to the Hardy–Weinberg equilibrium, there should be approximately 91 E8SJM homozygotes in the German population. Furthermore, taking into account the other potential mutations, they estimated that the population prevalence of CESD (including homozygotes and compound heterozygotes) in neonates would be approximately 25 cases/million.10
It has also been suggested that there is significant underdiagnosis because known cases seem to be few relative to gene frequency data that suggest a much higher incidence should be observed.11
The frequency of LIPA gene mutations varies by ethnic group. In Iranian Jews in the Los Angeles area, a mutation causing WD (p.G87V) has been shown to have a heterozygous gene frequency indicating that as many as 1 in 4200 live births in this community could be affected by WD.12 Whether this is due to a founder effect or is true of wider Middle Eastern groups needs further study.
The structural gene for hLAL maps to chromosome 10q23.2 has 10 exons and is about 36.5 kb in size. The 5′ flanking region is G+C-rich and has characteristics of a ‘housekeeping’ gene promoter. The full-length cDNA encoding hLAL is about 2.6 kb in length with 1200 nucleotides in the coding sequence.13 This produces a protein containing 399 amino acids with some variants in cotranslational cleavage sites meaning that the final protein differs between fibroblasts and liver, having 372 or 378 amino acids in the mature protein.14 ,15
A wide variety of mutations associated with LAL deficiency have been reported (table 1). Mutations associated with WD are associated with it either in homozygous or compound heterozygous individuals; also, CESD has been shown in some compound heterozygote patients who have one ‘WD’ mutation.16 Some mutations, for example, the exon 8 splice variant result in partial LAL deficiency, although 97% of the time a short inactive enzyme is produced, 3% of the time the correct version results, giving approximately 3% normal enzyme activity.4
As stated above, the mutated gene is on chromosome 8 and the disease occurs in homozygotes and compound heterozygotes and is therefore an autosomal recessive condition. No gender-specific patterns of disease have been described.
Wolman's disease in infants: cytoplasmic lipid accumulations can be identified in skin biopsies and there are also heavy accumulations in lymph nodes, spleen, adrenal glands, liver and gut.30 Infants with Wolman's disease may have intractable diarrhoea and severe malabsorption. In these cases, intestinal villi can be severely distorted and may be club-shaped due to infiltration of foam cells into the lamina propria.31 In some cases, the diagnosis has been made on intestinal biopsy specimens.32 ,33 Seen through the electron microscope, the microvilli are markedly shortened and irregular and may have severely impaired disaccharidase activity.31 Adrenal necrosis and calcification have been observed in some cases.34
CESD in adults: in rat models of CESD, lipid droplets and crystals are evident in hepatocytes and seen through the electron microscope, these are surrounded by a limiting membrane.35 There are also collections of foamy Kupffer cells filled with storage vacuoles,36 which form islets and are associated with desmin-postive Ito cells.35 These cells stain readily with periodic acid Schiff reagent and aldehyde fuchsin.37 In humans, there may be moderate intensity fat storage in cells associated with high-level receptor-mediated LDL endocytosis (hepatocytes and adrenal cortical cells), and also in Leydig cells in the seminiferous tubules.38 Histiocytes in gut, lymph nodes, spleen, bone marrow, lung or liver can be variably affected with some showing no evidence of storage, ranging to others with extreme lysosomal expansion and cholesteryl ester crystal formation.38 Bile duct epithelial cells may also show fat accumulation.37 There may also be signs of hepatic fibrosis in the periportal region, which may develop to cirrhosis.36 ,37 Hepatic cholesterol content may be 100–200× that of a normal liver39 and analysis of the fat content shows that it has a higher proportion of cholesterol linoleate [C18–2] (41%), lower proportion of cholesterol oleate [C18–1] (33%) and normal proportions of cholesterol palmitate [C16–0] (14%) when compared with controls.39
Due to the accumulation of fat in the intestine in WD, there are ultrasonographic and CT identifiable features.32 ,40 ,41 ,42 Specifically, the liver is enlarged with decreased CT density but normal US echogenicity; bowel loops are thickened and there is adrenal calcification. Contrast-enhanced CT demonstrates hepatosplenomegaly but there is low attenuation in the liver, which is indicative of excessive fat storage.43 In unenhanced T1-weighed MRI images, the adrenal glands have high signal intensity with subtle small foci of decreased intensity corresponding to calcified patches; the signal intensity of the spleen may be increased due to fat deposition; and retroperitoneal lymphadenopathy may be demonstrated.43 Ascites, adrenal calcification and thickened bowel loops may be identified by ultrasound.2 ,3
These features are present in infant onset of the disease but are not characteristic of CESD.
Biochemical testing and diagnosis
Diagnosis of LAL deficiency is possible using peripheral lymphocytes or cultured fibroblast cells,44 where activity of the enzyme is significantly decreased. There is no role for assay of serum enzymes (ie, serum acid phosphatase). Activity can been measured by measuring the lipase activity with 14C-triolein, 14C-cholesteryl oleate or 4-methyl-umbelliferyl-oleate.45 By studying the catabolic turnover of radiolabelled cholesteryl oleate in intact cells, lower in situ residual activity has been shown in cells with a C→T substitution at position 233, which induces a premature stop codon, than in cells having the His274 → Tyr substitution.46 14C-cholesteryl oleate and 4-methy-umbelliferyl-oleate have been used for prenatal diagnosis in cultured chorionic villus cells.47 ,48
Alternatively, again using live cells a fluorimetric technique where pyrinemethyl laurate (PMLes) is administered to cultured lymphoblastoid cells has been developed.49 ,50 PMLes hydrolysis by acid lipase can be followed directly in a spectrofluorometer because of the very high fluorescence emission of pyrene-methanol at 378 nm (monomeric form) in aqueous medium, whereas the substrate has practically no monomeric emission at 378 nm but emits only at 475 nm (excimeric form). In an alternative procedure, the enzymatic reaction can be determined after partition of the reaction mixture in a biphasic system of heptane and aqueous ethanol; the residual undegraded substrate partitioned into the upper heptane phase and the fluorescence of the product (ie, pyrene-methanol) is read in the lower aqueous-ethanolic phase, at 378 nm. PMLes is hydrolysed in extracts of normal lymphoblasts and fibroblasts by at least two lipases, one acidic lipase (pH 4.0) and a second more neutral enzyme (pH 6.5). The acidic lipase activity is practically absent in lymphoblasts and fibroblasts from WD or CESD.49 The two separate pathways of intracellular degradation of PMLes are mediated by lysosomal and extra-lysosomal hydrolases. PMLes incorporated into LDL is taken up by normal lymphoblastoid cells through the apolipoprotein-B/E-receptor-mediated pathway and degraded in the lysosomal compartment, resulting in a degradation block in Wolman cells. If PMLes dissolved in 2% dimethyl sulfoxide is added directly to the culture medium, its hydrolysis in lymphoblastoid cells from controls and from patients affected with WD, neutral lipid storage disease or familial hypercholesterolaemia is similar, indicating a pathway dependent on non-lysosomal enzyme, which is not deficient in Wolman cells.50
A recent advance in the biochemical assessment of LAL has been the development of a dried blood spot assay: Total acid lipase activity is measured using a 4-methyl-umbelliferyl palmitate substrate. Activity is then also measured with Lalistat 2 (a selective inhibitor of LAL) and the LAL activity is the difference between the two results.51
Identification of patients with CESD may be difficult because it is a rare disease and therefore may not be recognised. Various reports suggest that patients with the condition may have low HDL-cholesterol and elevated liver enzymes (Alanine Aminotransferase (ALT)).
Low HDL is not always present but is a usual finding: Lohse et al 16 showed that of two patients with CESD, although one had HDL-cholesterol=0.65 mmol/L, the other had HDL-cholesterol=0.96 mmol/L; Levy et al 52 reported a patient with HDL-cholesterol 0.67 mmol/L, which increased to 0.79 mmol/L with lovastatin treatment. Tadiboyina et al 53 reported a patient with HDL-cholesterol 0.46 mmol/L.
A study investigating whether routine laboratory data (HDL ≤ 0.8 mmol/L, ALT ≥ 60 IU/L) has recently begun. If successful, this may provide a route to assist in identification of patients who may benefit from treatment.54
WD has been treated successfully by haematopoietic cell transplantation using unrelated umbilical cord blood cell55 ,56 or bone marrow transplantation.57 This has been very successful with normalisation of end organ function and survival for 11 years being reported, compared with death in the first year of life being typical. Successful treatment results in resolution of diarrhoea within weeks of engraftment, as pathological lipid storage in the gut is restored to normality. Liver function also normalises and in one patient adrenal function has been reported to be normal. Stem cell treatment has not been used for patients with CESD.
In some patients with CESD, treatment with lovastatin has been reported:58 ,59 ,60 In a sibling pair, lovastatin resulted in a significant fall in serum cholesterol and resolution of hepatomegaly in one sibling, but in the other, response was not as good. The effect of lovastatin therapy has been assessed by scanning and has been proven to reduce but not normalise hepatic lipid content and similarly to reduce hepatic volume.52
Other treatment options have been used, including Simvastatin,24 ,61 Lovastatin and Ezetimibe,53 Simvastatin and Cholestyramine,62 Lovastatin and Cholestyramine.63 Treatment with these agents has been shown to decrease total serum cholesterol and increase HDL-cholesterol, which is important because it reduces cardiovascular disease risk, but the lipid accumulated in the liver is not normalised, so the risk of cirrhosis and liver failure persists. It is for this reason that other treatment options are considered necessary.
A gene transplant method to restore normal LAL function in mice has been tested:66 A first-generation adenoviral vector was used to transfect lal(−/−) mice with human LAL cDNA. Compared with controls, the treated mice showed increased hepatic LAL activity, decreased hepatomegaly and normalisation of histopathological features at 20 days after injection. The effectiveness of this method has also been demonstrated in fibroblast cells from a patient with WD.67
As an alternative to gene therapy, direct enzyme replacement can be used. Early experiments used cross-linking of Pseudomonas cholesteryl esterase to either porcine insulin or LDL particles to mediate absorption of enzyme into cells.68 It is now well recognised that mannose-6-phosphate is attached to lysosomal enzymes manufactured in the Golgi complex.69 These residues bind to mannose-6-phosphate receptors (MPR) and MPR-ligand complexes interact with the heterotetrameric complex of the AP1 clathrin adaptor complex70 where they are concentrated in tubular structure that help form with early endosomes.71 When these endosomes mature they merge with lysosomes, thus delivering the enzyme to the correct cellular compartment. Although this is an intracellular mechanism, the same process can be used to deliver extracellular mannose-labelled enzyme to lysosomes.72 ,73 ,74 This method has been used to successfully treat lal(−/−) mice. Just 10 injections of enzyme normalised hepatic colour, decreased liver weight (50%–58%), and diminished hepatic cholesterol and triglyceride storage.72 A direct enzyme replacement drug (SBC-102; Sebelipase Alfa) has been developed by Synageva Biopharma (Lexington, USA) and human trials are currently underway.75 Two phase II trials in Fabry disease have demonstrated that this method is a safe and effective way of delivering treatment.76 ,77 Phase I and safety data for acid lipase replacement therapy have demonstrated a rapid and sustained decrease in serum transaminases, an initial increase in serum LDL-cholesterol followed by significant decreases below baseline levels, and an increase in HDL-cholesterol.78 No major safety issues were identified and patients did not develop antibodies to LAL.
CESD is a rare disease that until recently was not easily treated. Consequently, there is no organised screening programme to identify patients with the disease. A potential treatment has now been developed that may allow effective treatment, similar to the treatment already listed in guidance for treatment of Gaucher's disease.79 Before this treatment was available, there was little incentive to screen for the disease but now attempts are being made to improve its identification,54 based on the known clinical features of decreased HDL-cholesterol and hepatic lipid accumulation leading to mild hepatic injury and elevated serum transaminases. Although this is currently a rarely identified disease, the identification and treatment of CESD due to LAL deficiency is likely to become important in the next few years.
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Competing interests Prof. Reynolds is currently in receipt of a project grant from SynaGeva Biopharma.
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