Enzymatic diagnosis of neuronal lipofuscinoses in dried blood spots using substrates for concomitant tandem mass spectrometry and fluorimetry

Abstract

Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative diseases predominantly in childhood that are characterized by psychomotor deterioration, epilepsy, and early death of patients. The NCLs analyzed in the present study are caused by defects of the specific enzymes, CLN1 (palmitoyl protein thioesterase 1; PPT1), CLN2 (tripeptidyl peptidase 1; TPP1), and CLN10 (cathepsin D). Specific and sensitive diagnostic assays of NCLs were the main goal of this study. They are of increasing importance, particularly since enzyme replacement therapy (ERT) for NCL2 has recently become available for clinical treatment, and ERTs for further NCLs are under development. Here, we report specific and sensitive determinations for CLN1, CLN2, and CLN10 on dried blood spots by tandem mass spectrometry using multiple reaction monitoring mass spectrometry (MRM‐MS). Identical substrates suitable for (i) fluorimetric determination of single enzymes and (ii) for MRM‐MS determination of multiple enzymes were synthesized by chemical coupling of alkyl‐umbelliferone building blocks with the corresponding peptidyl‐substrate groups recognized by the target enzyme. Enzymatic determinations were performed both by fluorimetry and MRM‐MS in patients with NCL1, NCL2, and NCL10 and showed good agreement in single assays. Moreover, duplex and triplex determinations were successfully performed for NCL1, NCL2, and NCL10. Specific peptidyl‐(4‐alkyl‐umbelliferone) substrates were also synthesized for mass spectrometric determinations of different cathepsins (cathepsins‐D, ‐F, and ‐B), to provide a differentiation of proteolytic specificities.

The LINCE Project: A Pathway for Diagnosing NCL2 Disease

“…Two of the patients in the present series were compound heterozygous, the first was homozygous, and the variants had already been described and classified as pathogenic ( 35 ). Of the 131 known NCL2 variants ( 34 ), c.509-1G>C and c.622C>T were found in all three patients. These variants are the two most frequent and account for 50% of disease-associated alleles (89% in Europe).…”

“…No false-negative results have been reported to date, and no NCL1 patients have been identified. More recently, tandem mass spectrometry (MSMS) assays for the determination of TPP1, PPT1 and cathepsin D activity in DBS have become available ( 30 – 34 ). Although MSMS assays yield higher analytical ranges than fluorometric assays ( 34 ), one of the advantages of our method is that it can measure PPT1 in only 5 h and TPP1 in 20 h, as opposed to the 45-h incubation reported by Lukacs et al ( 26 ) and similar to LC-MS/MS multiplex assays ( 34 ).…”

“…More recently, tandem mass spectrometry (MSMS) assays for the determination of TPP1, PPT1 and cathepsin D activity in DBS have become available ( 30 – 34 ). Although MSMS assays yield higher analytical ranges than fluorometric assays ( 34 ), one of the advantages of our method is that it can measure PPT1 in only 5 h and TPP1 in 20 h, as opposed to the 45-h incubation reported by Lukacs et al ( 26 ) and similar to LC-MS/MS multiplex assays ( 34 ). Efforts are currently being made in our laboratory to measure both TPP1 and PPT1 in the shortest time possible using mass spectrometry, because are the most commons.…”

Neuronal Ceroid Lipofuscinosis: The Multifaceted Approach to the Clinical Issues, an Overview

“…Advances in NCL genetics were accompanied by new opportunities to establish diagnosis at a biochemical level; enzymatic assays became available for four lysosomal enzymes: CTSD, CTSF, PPT1, and TPP1 ( 53 – 55 ). The gold standard for diagnosis is to require genetic confirmation by detecting both disease causing alleles.…”

“…Moreover, lysosomal dysfunction and dysregulated autophagy which are observed in CLN3 and CLN6 disease (49,50) are also seen in most forms of late-adulthood neurodegeneration (51,52). Advances in NCL genetics were accompanied by new opportunities to establish diagnosis at a biochemical level; enzymatic assays became available for four lysosomal enzymes: CTSD, CTSF, PPT1, and TPP1 (53)(54)(55). The gold standard for diagnosis is to require genetic confirmation by detecting both disease causing alleles.…”