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

JIMD Reports

Andrej Surkov¹  Olga Gundobina¹  Tatiana Strokova²  Nikolay Vlasov³  Ekaterina Brzhozovskaya⁴  Alexander Pushkov⁵  Kirill Savostianov⁵  Nikolay Mayanskiy⁶ 

[1] Department for gastroenterology National Medical Research Center for Children's Health Moscow Russia;Department for pediatric gastroenterology, hepatology and nutrition Federal Research Center for Nutrition and Biotechnology Moscow Russia;Department for pediatrics Filatov's Children Hospital, No.5 Saint‐Petersburg Russia;Laboratory department National Medical Research Center for Children's Health Moscow Russia;Laboratory for molecular genetics and cellular biology National Medical Research Center for Children's Health Moscow Russia;Russian Children Clinical Hospital Pirogov Russian National Research Medical University Moscow Russia;

关键词: cholesterol ester storage disease;    Lalistat‐2‐independent assay;    lysosomal acid lipase;    Wolman disease;   

DOI  :  10.1002/jmd2.12050

来源: DOAJ

【 摘 要 】

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‐AUC1h) 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.

【 授权许可】

Unknown