Newborn screening for lysosomal storage disorders.

Lysosomal storage disorders (LSD) represent a group of over 40 distinct genetic diseases with a total incidence of approximately 1:7,000 births. Bone marrow transplantation and enzyme replacement therapy are currently in use for the treatment of some disorders and new forms of enzyme and gene replacement therapy are actively being researched. The effectiveness of these therapies, particularly for the LSD involving the central nervous system and bone pathology, will rely heavily upon the early diagnosis and treatment of the disorder, before the onset of irreversible pathology. In the absence of a family history the only practical way to detect these disorders will be by a newborn screening program. One common feature of these disorders is an increase in the number and size of lysosomes within the cell from approximately 1% to as much as 50% of total cellular volume. Associated with this, is a corresponding increase in some lysosomal proteins. We propose that the measurement of one or more of these proteins in blood spots taken from Guthrie cards, will form the basis of a newborn screening program, for the detection of all LSD. We have identified a number of lysosomal proteins as potential markers for LSD. The level of these proteins has been determined in blood spots taken from Guthrie cards and in plasma samples from over 300 LSD affected individuals representing 25 disorders. Based on these results we have proposed a strategy for a newborn screening program involving a two tier system, utilizing time resolved fluorescence immunoquantification of the protein markers in the first tier, followed by tandem mass spectrometry for the determination of stored substrates in the second tier assays.

Recruitment and activation of SHP-1 protein-tyrosine phosphatase by human platelet endothelial cell adhesion molecule-1 (PECAM-1). Identification of immunoreceptor tyrosine-based inhibitory motif-like binding motifs and substrates.

Stimulation of platelet aggregation leads to tyrosine phosphorylation of a number of receptors and signaling molecules including platelet endothelial cell adhesion molecule-1 (PECAM-1). In this report, we demonstrate that both protein-tyrosine phosphatases SHP-1 and SHP-2 physically associate with different kinetics of assembly with tyrosine-phosphorylated human PECAM-1 during integrin alphaIIbbeta3-mediated platelet aggregation. Peptido-precipitation analysis revealed that tyrosine-phosphorylated peptides encompassing residues 658-668 and 681-691 of PECAM-1 bound specifically to both protein-tyrosine phosphatases SHP-1 and SHP-2. We further show that the association of SHP-1 with PECAM-1 occurs through the direct interaction of the src homology region 2 domains of SHP-1 with two highly conserved phosphotyrosine binding motifs within PECAM-1 having the sequences NSDVQpY663TEVQV and DTETVpY686SEVRK (where pY represents phosphotyrosine). In vitro dephosphorylation experiments using phosphotyrosyl PECAM-1 peptides encompassing either Tyr-663 or Tyr-686 revealed induction of SHP-1 catalytic activity, suggesting that PECAM-1 serves as a SHP-1 substrate. Surface plasmon resonance studies reveal that recombinant SHP-2 binds PECAM-1 phosphopeptides with 5-fold higher affinity than recombinant SHP-1. These data suggest that in hematopoietic cells such as platelets, PECAM-1 cellular signaling is regulated by the selective recruitment and activation of two distinct protein-tyrosine phosphatases, SHP-1 and SHP-2, via a common immunoreceptor tyrosine-based inhibitory-like motif.

Evaluation of the lysosome-associated membrane protein LAMP-2 as a marker for lysosomal storage disorders.

For many lysosomal storage disorders, presymptomatic detection, before the onset of irreversible pathology, will greatly improve the efficacy of current and proposed therapies. In the absence of a family history, presymptomatic detection can be achieved only by a comprehensive newborn screening program. Recently we reported that the lysosome-associated membrane protein LAMP-1 was increased in the plasma from approximately 70% of individuals with lysosomal storage disorders. Here we report on the evaluation of a second lysosome-associated membrane protein, LAMP-2, as a marker for this group of disorders. The median concentration of LAMP-2 in the plasma of healthy individuals was 1.21 mg/L, fourfold higher than the median LAMP-1 concentration (0.31 mg/L). LAMP-2 was increased in >66% of patients with lysosomal storage disorders, and the increases coincided with increased LAMP-1 concentrations. The reference intervals for LAMP-1 and LAMP-2 in blood spots taken from newborns were 0.20-0.54 mg/L (n = 1600) and 0.95-3.06 mg/L (n = 1600), respectively. A high correlation was observed between the concentrations of LAMP-1 and LAMP-2 in both control and affected individuals. The higher concentrations of LAMP-2, relative to LAMP-1, in plasma make LAMP-2 an attractive marker; however, the final selection will be dependent on the availability of new diagnostic markers and their ability to detect disorders currently not identified by LAMP-2.

Determination of the surface structure autocorrelation function for rough deposits of magnesium.

Autocorrelation functions for rough surfaces of evaporated opaque magnesium deposits are determined from electron microphotographs of the shadowed-surface carbon replicas. These microphotographs are analyzed, using a data-processing system microdensitometer. It is shown that autocorrelation functions do not have a Gaussian form, as is usually assumed.