Short-incubation mass spectrometry assay for lysosomal storage disorders in newborn and high-risk population screening

Abstract

The interest in early detection strategies for lysosomal storage disorders (LSDs) in newborns and high-risk population has increased in the last years due to the availability of novel treatment strategies coupled with the development of diagnostic techniques. We report the development of a short-incubation mass spectrometry-based protocol that allows the detection of Gaucher, Niemann-Pick A/B, Pompe, Fabry and mucopolysaccharidosis type I disease within 4h including sample preparation from dried blood spots. Optimized sample handling without the need of time-consuming offline preparations, such as liquid-liquid and solid-phase extraction, allows the simultaneous quantification of five lysosomal enzyme activities using a cassette of substrates and deuterated internal standards. Applying incubation times of 3h revealed in intra-day CV% values ranging from 4% to 11% for all five enzyme activities, respectively. In a first clinical evaluation, we tested 825 unaffected newborns and 16 patients with LSDs using a multiplexed, turbulent flow chromatography-ultra high performance liquid chromatography-tandem mass spectrometer assay. All affected patients were identified accurately and could be differentiated from non-affected newborns. In comparison to previously published two-day assays, which included an overnight incubation, this protocol enabled the detection of lysosomal enzyme activities from sample to first result within half a day.

Lentivector Iterations and Pre-Clinical Scale-Up/Toxicity Testing: Targeting Mobilized CD34 + Cells for Correction of Fabry Disease

“…The prevalence of males with Fabry disease ranges from 1:17,000 to 1:117,000 in the Caucasian population 4, 5. Recent newborn screening studies have, however, indicated a much higher incidence of this disorder because patients with later onset and milder forms of Fabry disease are found even more commonly than expected, suggesting that they are possibly underdiagnosed 6, 7, 8, 9, 10, 11, 12, 13…”

Screening for late-onset Pompe disease in Finland

“…Thus, accurate and early diagnosis also in late-onset disease is needed [5]. The measurement of GAA activity in dried blood spots (DBS) has been successfully used to screen for Pompe disease, and is suggested to be the optimal initial diagnostic test procedure [6,7].…”

Pompe Disease

“…21-23 As a result, multiplex newborn screening for Pompe disease and other lysosomal storage disorders using fluorometric, digital microfluidic and tandem mass spectrometry based GAA enzyme activity assays had been developed. 24-27 In addition to qualitative and quantitative assessments of the disease burden, and clinical measures of the impact of Pompe disease on various affected systems, urinary glucose tetrasaccharide (Glc4), a biomarker of glycogen storage with 94% sensitivity and 84% specificity for Pompe disease, is frequently used in monitoring the response of patients to enzyme replacement therapy and as an adjunct to acid α-glucosidase activity measurements. 28 Also, in addition to the traditional 1-dimensional thin layer chromatography (TLC) for urine oligosaccharide analysis, a new MALDI–time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry based assay of urinary free oligosaccharides useful for the diagnosis of Pompe disease and other lysosomal storage diseases is now available.…”