WP02: Automated enzyme testing

WP leader

Herbert Korall, PhD
Zentrum für Stoffwechseldiagnostik (ZfS)

 

Michael Przybylski, PhDSteinbeiss GmbH & Co KG

WP Summary

 

Objectives

  • To establish an automated enzyme testing methodology for CLN1, CLN2, and CLN10 diseases in dry blood spots using tandem mass spectrometry, in order to obtain a tool for easy rapid low-cost diagnosis worldwide.
  • To perform cost-effective high-throughput enzyme testing for CLN1, CLN2, and CLN10 diseases in order to estimate prevalence and measure birth incidence of these types of NCL in selected geographical areas.


Establishment of an automated enzyme testing methodology for CLN1, CLN2, and CLN10 diseases in dry blood spots using tandem mass spectrometry


Transfer of existing enzymatic fluorometric assays to tandem mass spectrometry (TMS)

 

At the beginning of the project, enzymatic fluorometric assays for determination of palmitoyl protein thioesterase 1 (PPT1 / CLN1), tripeptidyl peptidase 1 (TPP1 / CLN2) and cathepsin D (CLN10) in dried blood spots had already been established by partner 01 and were regularly used in the NCL diagnostic service of partner 01 (Lukacs et al., 2003). In order to establish an automated enzyme testing methodology in the laboratory of SME partner 07, this technique was transferred to tandem mass spectrometry (TMS).

The enzymatic activity assays for CLN1 and CLN2 were carried out according to the procedures published by Lukacs et al. The sample workup was altered due to the requirements for tandem mass spectrometry. For quality control, dry blood spot samples derived from patients with genetically confirmed diagnosis of CLN1 or CLN2, respectively, were used. Samples were tested in parallel by partner 07 using the newly developed TMS-based method as well as by partner 01 using the original fluorometric assay. Table 1 gives an overview of the results obtained by each method. At a qualitative scale, all patients could be clearly assigned to their respective disease using TMS. However, quantitative data differed between the two methods. TMS and fluorescence assays both lead to the same results in respect to the diagnosis of CLN1 or CLN2 deficiency. Affected patients could be clearly distinguished from not affected persons or healthy carriers.

 

Table 1:  Quantitative data of known patients affected with CLN1 or CLN2 (LOD=Limit of Detection)


 

When attempting to transfer enzymatic fluorometric assays to tandem mass spectrometry (TMS), SME partner 07 came across several challenges:

 

i) No method for fluorometric measurement of cathepsin D (CLN10) activity in dried blood spot was published in the literature so far. During the first reporting period, partner 07 did not succeed in establishing a selective method using two different already commercially available substrates and different assay conditions. None of the methods could clearly distinguish between positive and negative controls, e.g. wild type and cathepsin D knock out mice. Moreover, gathering samples from patients with cathepsin D deficiency was difficult, due to the fact of the rareness of this specific disease and of the early death within weeks after birth of most affected patients. Affected and unaffected mice showed higher amounts of reaction product in comparison to healthy human samples, which may be justified with the different metabolisms.

ii) Use of the original fluorometric substrates in mass-spectrometry-based assays required frequent cost- und time-intensive cleaning of the mass spectrometer which increased diagnostic costs per sample significantly. Consequently, costs for the initially intended screening of a high number of anonymous dry blood spots collected from newborn-screening programs became too high (>200 000 €).

Taken together, these problems led to the following change in the work program for the current reporting period which had been agreed with the EC scientific officer as part of amendment No. 2 of the original DoW:

The consortium agreed that the use of completely newly developed substrates for all three mass-spectrometry based enzyme activity assays (CLN1, CLN2, and CLN10) was necessary. This led to the addition of partner 13 (StC) to the consortium who recently had successfully developed such substrates for CLN1, CLN2, and CLN10 enzyme activity assays which are suitable for both – fluorometric and MS-based assays  - without requiring required frequent cost- und time-intensive cleaning of the mass spectrometer. Moreover, the consortium agreed, that the successful application of these new substrates should lead to the development of a MS-based triplex enzyme assay allowing measurement of enzyme activities of CLN1, CLN2, and CLN10 in the same assay. This would render the assay more time- and cost-effective.

 

 

Development of a MS-based multiplex array for CLN1, CLN2 and CLN10

 

The development of a MS-based specific and highly sensitive clinical diagnostics triplex assay for CLN1, CLN2, and CLN10 (cathepsin D) in dry blood spots (DBS) was successfully performed taking several steps:

1. Successful adaptation of newly developed specific substrates based as internal standards suitable for triplex assay 

2. Mass spectrometric (MS-MRM) single determinations of enzyme activities, validation by fluorimetric determination

3. Development of a mass spectrometric triplex assay for CLN1, CLN2, CLN10 by MS-MRM using the new substrate derivatives

4. Evaluation of triplex assay by determination of positive control patient samples (Fig. 2)

 

 

Fig. 2. Tripeptidyl peptidase activity determined by MS-MRM for CLN2 patients and healthy donors.


Performance of cost-effective high-throughput enzyme testing for CLN1, CLN2, and CLN10 diseases in order to estimate prevalence and measure birth incidence of these types of NCL in selected geographical areas


During the first reporting period, the original approach to measure birth incidence figures for specific NCL forms (CLN1, CLN2, CLN10) was to screen a high number of anonymous dry blood spots collected from newborn-screening programs. As the use of the original fluorometric substrates in mass-spectrometry-based assays required frequent cost- und time-intensive cleaning of the mass spectrometer the diagnostic costs per sample increased significantly. Consequently, costs for the initially intended screening of a high number of anonymous dry blood spots collected from newborn-screening programs became too high (>200 000 €). These problems led to the following change in the work program agreed with the EC scientific officer as part of amendment No. 2:

Instead of using a large number of anonymous dry blood spots, the consortium suggested to perform targeted population screening in order to estimate incidence figures of CLN1, CLN2, and CLN10 disease: Due to the above mentioned challenges and the necessity to include a new partner into the consortium, the new triplex enzyme assay using the new substrates had only been available since end of month 35 of the project. Therefore statistical numbers from targeted population screening are still under way. Further results are explained in WP03.

 

Conclusion


In WP2, a mass-spectrometry based multiplex enzyme activity assay has successfully been developed which is able to measure enzyme activities for CLN1, CLN2, and CLN10 disease in parallel.