Biomedical Scientist, doctorate in Bioengineering. Determined to contribute to a world in which Healthcare and Innovation are accessible to everyone.
Testing for diagnosis of Lysosomal Acid Lipase Deficiency (LAL-D) is conducted once the disease is suspected. This usually occurs in individuals presenting with growth failure (in the case of infantile LAL-D), hepatomegaly, liver dysfunction and abnormal lipid profile.1,2
Preliminary testing consists of a lipid panel which typically involves the measurement of serum concentrations of total, low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterols, along with triglycerides.1–3 Once suspected, LAL-D can usually be diagnosed by 2 different tests: identification of pathogenic variants in the LIPA gene and measurement of LAL enzymatic activity in peripheral blood leukocytes, fibroblasts, hepatocytes or dried blood spots (DBS).1,2,4 The histopathological analysis of liver biopsies and radiological/imaging techniques are useful in supporting the diagnosis of LAL-D, even though such methods are not considered diagnostic per se.2 Initial assessment tests may also include detection of liver injury and kidney function biomarkers, as well as a full hematologic evaluation.3
Serum lipid concentrations are almost always abnormal in patients with LAL-D , though a diagnosis of the disease is not excluded by normal serum lipid levels.1,4 Hoffman and colleagues reviewed the lipid values in 33 patients with LAL-D.1 Triglycerides, total cholesterol and LDL were found significantly elevated in 71%, 88% and 100% of cases, while HDL was significantly reduced in 96% of individuals. Clinical laboratories typically report the LDL concentration as a calculated value using the Friedewald equation (LDL = total cholesterol – HDL – triglycerides, with all values in mg dL-1) as long as triglyceride levels are below 400 mg dL-1 (4.52 mmol/L).5,6
In case of other cardiovascular risk factors or a family history of cardiovascular disease, advanced lipid testing should be conducted, which measures specific subpopulations of lipoproteins and apolipoproteins including lipoprotein(a), apolipoprotein A (apoA), apolipoprotein B (apoB), as well as lipoprotein particle composition.3,7
Approaches for genetic testing may include single-gene testing (sequence analysis of the LIPA gene), use of a multigene panel (includes LIPA and other genes of interest for differential diagnosis) and comprehensive genomic testing (exome sequencing, genome sequencing, mitochondrial sequencing).1
Sequence analysis detects pathogenic variants, which may include small intragenic deletions/insertions as well as missense, nonsense and splice site variants.1 If only one pathogenic variant is identified, gene-targeted analysis of intragenic deletions or duplications should be considered. Methods used may include quantitative polymerase chain reaction (qPCR), long-range PCR, multiplex ligation-dependent probe amplification (MLPA) and gene-targeted microarray.1,8–10 To date, over 120 variants in the LIPA gene have been identified for LAL-D.9–11
Comprehensive genomic testing can be considered if serial single-gene testing and/or use of a multigene panel fail to confirm a diagnosis in an individual presenting features of LAL-D.1
Measuring LAL Activity
Enzymatic LAL activity can be measured biochemically in peripheral blood leukocytes, hepatocytes, skin fibroblasts or dried blood spots (DBS) that have been promptly transported and properly stored.2 Enzymatic activities for peripheral leukocytes or cultured fibroblasts from a cohort of 135 LAL-D patients were described to range from “undetectable” to 16% of normal LAL activity, with most patients exhibiting activity values between <1% and 10%.12 Substrates used in these assays, such as 4-nitrophenyl palmitate, are not specific for LAL and may react with other lipases, thereby precluding direct comparisons of the residual LAL activities among patients.
On the other hand, the accuracy of DBS testing can be substantially improved by determining LAL activity using 4-methylumbel-liferyl-palmitate as the enzyme substrate together with a highly specific inhibitor of LAL, Lalistat 2.13 LAL activity is calculated by comparing total lipase activity with and without the presence of Lalistat 2. DBS testing is able to distinguish normal individuals from CESD homozygotes and heterozygotes, has several advantages regarding sample size and stability and, thus, became the standard tool in academic and commercial labs worldwide to assess LAL activity.
Histopathological analysis of tissues affected by LAL-D using routine stains, such as hematoxylin and eosin (H&E), has been useful since the first reported case of the disease was studied by Wolman’s team in 1956.14 Using H&E, lipid-laden foam cells were identified in xanthomatous tissue of several organs, including the intestine, lymph nodes, thymus and adrenals. The positive Liebermann-Burchardt reaction, the sustainability with Sudan and the double refractivity, as well as the solubility characteristics and the absence of staining by the periodic acid-Schiff technique, showed that the lipids contained within these cells were predominantly cholesterol. H&E also allowed the visualization of areas of calcification in the adrenal glands.
Nowadays, histologic analysis to support the diagnosis of LAL-D mainly aims at detecting the presence of microvesicular steatosis, fibrosis/cirrhosis and ceroid-laden macrophages.3 However, such features are not exclusive to LAL-D. For example, microvesicular steatosis can also be visualized in non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH) or cryptogenic liver disease.12 Hůlková and Elleder developed a method for distinctive histological diagnosis of LAL-D using paraffin-embedded tissue samples obtained from liver biopsies.15 The method is based on the immunostaining identification of lysosomal lipid accumulation markers, namely lysosomal-associated membrane protein 1 (LAMP1), LAMP2, lysosomal integral membrane protein 2 (LIMP2) and lysosomal luminal cathepsin D. Additional diagnostic traits included auto-fluorescent detection of ceroid induction in storage macrophages, the absence of lipopigment in hepatocytes and identification of Maltese cross-type birefringence-associated stored liquid crystals of cholesteryl esters (the latter in unfixed biopsy samples).
X-rays were used by Abramov et al. in 1956 to examine adrenal calcifications.14
Hepatic magnetic resonance represents a non-invasive approach to characterize and monitor the hepatic lipid signature in LAL-D patients.16
Hepatic fibrosis can be assessed noninvasively by transient elastography or other imaging methods that measure liver stiffness, like acoustic radiation force impulse imaging and shear-wave transient elastography.3 Transient elastography using FibroScan® is a first-line method for the detection of fibrosis and cirrhosis in non-obese patients.17
Hepatic, Renal and Hematological Evaluation
Hepatic evaluation can also include the biochemical examination of indicators of liver injury, particularly serum levels of alanine aminotransferase (ALT), aspartate aminotransaminase (AST) and γ-glutamyltransferase (GGT).3 In turn, renal evaluation may include testing for kidney function biomarkers, such as blood urea nitrogen (BUN), serum creatinine (Cr) and estimated glomerular filtration rate (eGFR).3
Hematologic testing typically comprises evaluation of complete, red and white blood cell counts, hemoglobin, hematocrit, platelet count, prothrombin time and international normalized ratio.3
1. Hoffman EP, Barr ML, Giovanni MA, Murray MF. Lysosomal acid lipase deficiency. 2015 Jul 30 [updated 2016 Sep 1]. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Mirzaa G, Amemiya A, editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993–2021.
2. Reiner Ž, Guardamagna O, Nair D, et al. Lysosomal acid lipase deficiency – an under-recognized cause of dyslipidaemia and liver dysfunction. Atherosclerosis. 2014;235(1):21-30. doi:https://doi.org/10.1016/j.atherosclerosis.2014.04.003
3. Kohli R, Ratziu V, Fiel MI, Waldmann E, Wilson DP, Balwani M. Initial assessment and ongoing monitoring of lysosomal acid lipase deficiency in children and adults: consensus recommendations from an international collaborative working group. Mol Genet Metab. 2020;129(2):59-66. doi:10.1016/j.ymgme.2019.11.004
4. Strebinger G, Müller E, Feldman A, Aigner E. Lysosomal acid lipase deficiency – early diagnosis is the key. Hepat Med. 2019;11:79-88. doi:10.2147/HMER.S201630
5. Jacobson TA, Ito MK, Maki KC, et al. National lipid association recommendations for patient-centered management of dyslipidemia: part 1-full report. J Clin Lipidol. 2015;9(2):129-169. doi:10.1016/j.jacl.2015.02.003
6. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6):499-502.
7. Chandra A, Rohatgi A. The role of advanced lipid testing in the prediction of cardiovascular disease. Curr Atheroscler Rep. 2014;16(3):394. doi:10.1007/s11883-013-0394-9
8. Fasano T, Pisciotta L, Bocchi L, et al. Lysosomal lipase deficiency: molecular characterization of eleven patients with wolman or cholesteryl ester storage disease. Mol Genet Metab. 2012;105(3):450-456. doi:https://doi.org/10.1016/j.ymgme.2011.12.008
9. Anderson RA, Bryson GM, Parks JS. Lysosomal acid lipase mutations that determine phenotype in wolman and cholesterol ester storage disease. Mol Genet Metab. 1999;68(3):333-345. doi:10.1006/mgme.1999.2904
10. Pisciotta L, Tozzi G, Travaglini L, et al. Molecular and clinical characterization of a series of patients with childhood-onset lysosomal acid lipase deficiency. retrospective investigations, follow-up and detection of two novel <em>LIPA</em> pathogenic variants. Atherosclerosis. 2017;265:124-132. doi:10.1016/j.atherosclerosis.2017.08.021
11. Carter A, Brackley SM, Gao J, Mann JP. The global prevalence and genetic spectrum of lysosomal acid lipase deficiency: A rare condition that mimics NAFLD. J Hepatol. 2019;70(1):142-150. doi:10.1016/j.jhep.2018.09.028
12. Bernstein DL, Hülkova H, Bialer MG, Desnick RJ. Cholesteryl ester storage disease: review of the findings in 135 reported patients with an underdiagnosed disease. J Hepatol. 2013;58(6):1230-1243. doi:https://doi.org/10.1016/j.jhep.2013.02.014
13. Hamilton J, Jones I, Srivastava R, Galloway P. A new method for the measurement of lysosomal acid lipase in dried blood spots using the inhibitor lalistat 2. Clin Chim Acta. 2012;413(15-16):1207-1210. doi:10.1016/j.cca.2012.03.019
14. Abramov A, Schorr S, Wolman M. Generalized xanthomatosis with calcified adrenals. AMA J Dis Child. 1956;91(3):282-286. doi:10.1001/archpedi.1956.02060020284010
15. Hůlková H, Elleder M. Distinctive histopathological features that support a diagnosis of cholesterol ester storage disease in liver biopsy specimens. Histopathology. 2012;60(7):1107-1113. doi:https://doi.org/10.1111/j.1365-2559.2011.04164.x
16. Thelwall PE, Smith FE, Leavitt MC, et al. Hepatic cholesteryl ester accumulation in lysosomal acid lipase deficiency: non-invasive identification and treatment monitoring by magnetic resonance. J Hepatol. 2013;59(3):543-549. doi:https://doi.org/10.1016/j.jhep.2013.04.016
17. Afdhal NH. Fibroscan (transient elastography) for the measurement of liver fibrosis. Gastroenterol Hepatol (NY). 2012;8(9):605-607. https://pubmed.ncbi.nlm.nih.gov/23483859
Reviewed by Michael Sapko, MD on 7/1/2021.