A kinetic assay of total lipase activity for detecting lysosomal acid lipase deficiency (LAL‐D) and the molecular characterization of 18 LAL‐D patients from Russia

Abstract

Laboratory diagnostics of lysosomal acid lipase deficiency (LAL‐D), a rare disorder associated with LIPA alterations, are based on the evaluation of LAL activity. In dry blood spots (DBS) submitted for LAL‐D diagnostics (the screening cohort) over a two‐year period or obtained from a cohort of retrospective LAL‐D patients, we measured: (1) LAL activity using a two‐reaction assay with 4‐methylumbelliferone palmitate (4‐MU‐Palm) and Lalistat‐2, a specific LAL inactivator; (2) total lipase (TL) activity by a 1‐hour kinetic 4‐MU‐Palm cleavage reaction (no Lalistat‐2). The TL activity was expressed as the area under the kinetic curve after 1 hour (TL‐AUC 1h ) of the reaction and presented as the median (min‐max). LAL activity was reduced in 30/537 individuals from the screening cohort, among which LIPA sequencing revealed six patients and one carrier. Overall, 16 (89%) individuals among six novel and 12 retrospective LAL‐D patients carried at least one c.894G>A mutation (six were homozygous). The TL‐AUC1h in nonLAL‐D specimens with normal LAL activity (n = 90) was unambiguously higher (9471 [4015‐23 585] RFU*h/punch) compared to LAL‐D patients, including six new and nine retrospective patients (1810 [357‐2608] RFU*h/punch). Importantly, in 13/15 examined nonLAL‐D specimens with reduced LAL activity the TL‐AUC1h was above a threshold of 2652 RFU*h/punch. Applying this threshold, the TL‐AUC1h index discriminated all LAL‐D patients (100% sensitivity) and 103/105 nonLAL‐D specimens (98% specificity). Given that there is no need for Lalistat‐2 and two parallel enzymatic reactions in conjunction with high sensitivity and specificity, the kinetic assay seems to be practical for LAL‐D screening. Synopsis Lysosomal acid lipase deficiency responsible for Wolman disease and cholesterol ester storage disease could be reliably detected using a kinetic assay of total lipase activity with a fluorogenic substrate.

Newborn screening for lipid disorders

“…Enzyme replacement therapy for Wolman disease with sebelipase alfa was recently approved by the FDA, which can be life saving for Wolman syndrome patients. Assessment of LAL activity on dried blood spots in at-risk populations has been successfully performed in both France [61] and Russia [62], which paves the way for potential newborn screening programs for LAL-D deficiency and Wolman disease, though additional validation and implementation are likely necessary.…”

Lysosomal Acid Lipase Deficiency: Genetics, Screening, and Preclinical Study

“…Mayanskiy et al reported the LAL activity of DBS in a Russian population [ 45 ]. In their study, the authors researched a sample of 537 LAL-D-suspected individuals undergoing clinical screening between June 2016 and July 2018.…”

“…Originally, Hamilton et al reported the inclusion of Lalistat-2 in DBS to quantify LAL enzyme activity [ 10 , 29 ]. However, Mayanskiy et al reported a kinetic assay of the LAL enzyme without Lalistat-2, showing 100% sensitivity of LAL-D-affected individuals ( n = 6) and near-complete sensitivity of non-LAL-D individuals (103 out of 105) [ 45 ]. As long as assay specificity is guaranteed and assay equipment is available, the latter assay can be useful.…”

“…In LAL-D, no newborn screening is currently performed. Based on the success of high-risk screening in Russia [ 45 ] and France [ 44 ], newborn screening for LAL-D is increasingly demanded.…”

Off-target effects of the lysosomal acid lipase inhibitors Lalistat-1 and Lalistat-2 on neutral lipid hydrolases

“…L1 or L2 have been used in >50 scientific studies to investigate defective lysosomal lipid hydrolysis in various cellular processes such as autophagy and lipolysis [ [18] , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] ]. In the clinical setting, L2 found its application to specifically measure LAL activity in human blood samples and cells with the goal of simpler and more cost-effective diagnosis of LAL-D [ [27] , [28] , [29] ]. L1 and L2 have been shown to competitively inhibit LAL activity without affecting the activities of the extracellular lipolytic enzymes human pancreatic lipase and bovine lipoprotein lipase [ 30 , 31 ].…”