Direct multiplex assay of lysosomal enzymes in dried blood spots for newborn screening

Abstract

Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source.

Methods: We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid alpha-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed.

Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approximately one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals.

Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated, and the anticipated reagent and supply costs are well within the budget limits of newborn-screening centers.

Fig. 1. Substrates, products, and internal standards for the five lysosomal enzyme assays

The masses of the products and internal standards are indicated. The ceramide products and internal standards ASM-P, ASM-IS, GALC-P, GALC-IS, ABG-P, and ABG-IS undergo CID to give the common imminium ion shown (m/z = 264). GLA-P, GLA-IS, GAA-P, and GAA-IS undergo neutral-loss CID to give four different secondary ammonium ions. Enzymatic reactions with DBS are shown by solid arrows, and CID is shown by dashed arrows.

Fig. 2. Inhibition of acid α-glucosidases by acarbose (top) and Ghavamiol (bottom)

●, recombinant GAA; ▼, PMN enzymes; ■, recombinant GAA + PMN enzymes. PMN enzyme activity was measured in a Triton X-100 lysate of PMNs. Assays were performed as described in Materials and Methods except that the incubation time was 70 min, with 2.3 ng of recombinant GAA and 1.3 μg of PMN protein used per assay.

Fig. 3. Amount of enzymatic reaction product vs assay incubation time

Each point is the mean (SD; error bars) of a triplicate assay, each carried out with one 2-mm DBS from a healthy adult per reaction tube. Assays as described in Materials and Methods except for the variation in the assay incubation time as indicated.

Fig. 4. Amount of enzymatic reaction product vs the number of 2-mm DBS

Assays were done with DBS from a healthy adult. Each point is the mean (SD; error bars) of triplicate assays carried out as described in Materials and Methods except for variation in the number of DBS as indicated.

Fig. 5. Activities of lysosomal enzymes in DBS obtained between 2000 and 2003, as measured using a single 5-mm DBS extracted with universal extraction buffer

Each box represents data obtained from 15 healthy adults. For each enzyme, data are arranged from 2000 to 2003 (left to right). All DBS were stored at 4 °C. The error bars indicate the range of values; and the boxes show the values between the 25th and 75th percentiles. Horizontal bars and ◆ in each box indicate the median and mean values, respectively.

Fig. 6. Enzyme activities in affected patients, heterozygotes, and healthy controls obtained with extracts of a 5-mm DBS

The number of individuals per group is indicated. GD, ABG activity in patients with Gaucher disease (1 infant, 2 juveniles, 3 adults); GC, ABG activity in heterozygous Gaucher adults; GHA, ABG activity in healthy adults; GHI, ABG activity in healthy infants; NPD, ASM activity in patients with Niemann–Pick disease types A and B (3 infants, 2 juveniles); NPC, ASM activity in heterozygous adults; NPHA, ASM activity in healthy adults; NPHI, ASM activity in healthy infants; KD, GALC activity in patients with Krabbe disease (1 infant, 2 juveniles, 1 adult, 5 of unknown age); KHA, GALC activity in healthy adults; KHI, GALC activity in healthy infants; PD, GAA activity in patients with Pompe disease (1 infant, 4 adults); PC, GAA activity in heterozygous adults; PHA, GAA activity in healthy adults; PHI, GAA activity in healthy infants; FD, GLA activity in patients with Fabry disease (3 juveniles, 2 adults); FC, GLA activity in heterozygous adults; FHA, GLA activity in healthy adults; FHI, GLA activity in healthy infants. DBS were stored at 4 °C except for the DBS obtained from GALC patients, which were stored at −20 °C.

Fig. 7. Enzymatic activities of affected patients, heterozygotes, and healthy controls obtained with a single 2-mm DBS per assay

Abbreviations are as in the legend for Fig. 6. DBS were stored at 4 °C except for the DBS obtained from GALC patients, which were stored at −20 °C.