Development of a newborn screening tool based on bivariate normal limits: using psychosine and galactocerebrosidase determination on dried blood spots to predict Krabbe disease.

PURPOSE: Newborn screening for Krabbe disease (KD) originated in New York State in 2006 but has proven to have a high false positive rate and low positive predictive value. To improve accuracy of presymptomatic prediction, we propose a screening tool based on two biomarkers, psychosine and galactocerebrosidase enzyme activity (GalC). METHODS: We developed the tool using measures from dried blood spots of 166 normal newborns and tested it on dried blood spot measures from 15 newborns who later developed KD, 8 newborns identified as "high risk" by the New York screening protocol but were disease-free at follow-up, and 3 symptomatic children with onset before 4 years of age. The tool was developed from the (1-10-6)100% prediction region of the natural logarithms of psychosine and GalC measures, assuming bivariate normality, and their univariate normal limits. RESULTS: Krabbe disease was predicted correctly for every patient who developed symptoms in infancy or early childhood. None of the high-risk patients were incorrectly identified as having early KD. CONCLUSION: Bivariate analysis of psychosine and GalC in newborn blood spots can accurately predict early Krabbe symptoms, control false positive rates, and permit presymptomatic treatment.

Large-scale study of clinical and biochemical characteristics of Chinese patients diagnosed with Krabbe disease.

Krabbe disease (KD) is a rare disease caused by the deficiency of ß-galactocerebrosidase. This study investigated 22 unrelated Chinese patients, including their clinical presentations, plasma psychosine levels and ß-galactocerebrosidase gene mutations. We found the late-onset form of KD present in 82% of the patients in our study, which was more prevalent than in patients from other populations. Plasma psychosine levels were elevated in KD, which were correlated with the severity of clinical presentations. Sanger sequencing identified 8 novel mutations, including 7 missense mutations, p.H253Y, p.S259L, p.P318L, p.F350V, p.T428A, p.L530P, p.G586D, and 1 splicing mutation, c.1251+1G>A. Quantitative real-time polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification identified a novel exon 12 and 14 deletion, separately. Next generation sequencing, applied at the final step, revealed 2 missense mutant alleles missed using Sanger sequencing. The most common mutation in Chinese population is p.P154H, which accounts for 20.5% of alleles. Consistent with the higher prevalence of the late-onset form of KD, missense mutations predominated in our study, different with the common mutation types in Europe and Japan. This work was the first large-scale study of Chinese KD patients describing their clinical, biochemical and genetic characteristics, which furthered our understanding of this classical neurological lysosomal storage disease.

A Comparison of Lysosomal Enzymes Expression Levels in Peripheral Blood of Mild- and Severe-Alzheimer's Disease and MCI Patients: Implications for Regenerative Medicine Approaches.

The association of lysosomal dysfunction and neurodegeneration has been documented in several neurodegenerative diseases, including Alzheimer's Disease (AD). Herein, we investigate the association of lysosomal enzymes with AD at different stages of progression of the disease (mild and severe) or with mild cognitive impairment (MCI). We conducted a screening of two classes of lysosomal enzymes: glycohydrolases (ß-Hexosaminidase, ß-Galctosidase, ß-Galactosylcerebrosidase, ß-Glucuronidase) and proteases (Cathepsins S, D, B, L) in peripheral blood samples (blood plasma and PBMCs) from mild AD, severe AD, MCI and healthy control subjects. We confirmed the lysosomal dysfunction in severe AD patients and added new findings enhancing the association of abnormal levels of specific lysosomal enzymes with the mild AD or severe AD, and highlighting the difference of AD from MCI. Herein, we showed for the first time the specific alteration of ß-Galctosidase (Gal), ß-Galactosylcerebrosidase (GALC) in MCI patients. It is notable that in above peripheral biological samples the lysosomes are more sensitive to AD cellular metabolic alteration when compared to levels of Aß-peptide or Tau proteins, similar in both AD groups analyzed. Collectively, our findings support the role of lysosomal enzymes as potential peripheral molecules that vary with the progression of AD, and make them useful for monitoring regenerative medicine approaches for AD.

Pilot study of newborn screening for six lysosomal storage diseases using Tandem Mass Spectrometry.

BACKGROUND: There is current expansion of newborn screening (NBS) programs to include lysosomal storage disorders because of the availability of treatments that produce an optimal clinical outcome when started early in life. OBJECTIVE: To evaluate the performance of a multiplex-tandem mass spectrometry (MS/MS) enzymatic activity assay of 6 lysosomal enzymes in a NBS laboratory for the identification of newborns at risk for developing Pompe, Mucopolysaccharidosis-I (MPS-I), Fabry, Gaucher, Niemann Pick-A/B, and Krabbe diseases. METHODS AND RESULTS: Enzyme activities (acid α-glucosidase (GAA), galactocerebrosidase (GALC), glucocerebrosidase (GBA), α-galactosidase A (GLA), α-iduronidase (IDUA) and sphingomyeline phosphodiesterase-1 (SMPD-1)) were measured on ~43,000 de-identified dried blood spot (DBS) punches, and screen positive samples were submitted for DNA sequencing to obtain genotype confirmation of disease risk. The 6-plex assay was efficiently performed in the Washington state NBS laboratory by a single laboratory technician at the bench using a single MS/MS instrument. The number of screen positive samples per 100,000 newborns were as follows: GAA (4.5), IDUA (13.6), GLA (18.2), SMPD1 (11.4), GBA (6.8), and GALC (25.0). DISCUSSION: A 6-plex MS/MS assay for 6 lysosomal enzymes can be successfully performed in a NBS laboratory. The analytical ranges (enzyme-dependent assay response for the quality control HIGH sample divided by that for all enzyme-independent processes) for the 6-enzymes with the MS/MS is 5- to 15-fold higher than comparable fluorimetric assays using 4-methylumbelliferyl substrates. The rate of screen positive detection is consistently lower for the MS/MS assay compared to the fluorimetric assay using a digital microfluidics platform.

The plasma activities of lysosomal enzymes in infants with necrotizing enterocolitis: new promising class of biomarkers?

BACKGROUND: Intestinal ischemia plays a major role in the pathogenesis of necrotizing enterocolitis (NEC). The diagnosis of intestinal ischemia would be highly desirable, as it is impossible to achieve with the current diagnostic regimes. Preliminary data from an animal NEC model indicate a possible correlation between the plasma activity of the lysosomal enzyme beta-glucosidase and intestinal ischemia. METHODS: In this case-control study the plasma activities of six different lysosomal enzymes were detected by high-performance liquid-chromatography tandem mass-spectrometry in 15 infants with NEC and compared to 18 controls. RESULTS: The plasma activities of ß-glucosidase (ABG), α-glucosidase (GAA), and galactocerebrosidase (GALC) were significantly higher in the NEC group compared with controls (ABG, p=0.009; GAA, p<0.001; GALC, p<0.001). GAA and GALC showed the highest diagnostic value with areas under the curve of 0.91 and 0.87. CONCLUSIONS: We identified GAA and GALC as new promising biomarkers for gut wall integrity in infants with NEC, and report first results on the plasma activity of ABG. The present study supports the hypothesis that the plasma activity of ABG might serve as a marker of intestinal ischemia in NEC. The identification of intestinal ischemia could facilitate early discrimination of infants at risk for NEC from infants with benign gastrointestinal disorders.

Does galactocerebrosidase activity predict Krabbe phenotype?

This study sought to determine whether galactocerebrosidase activity is predictive of Krabbe onset age, or of survival from onset when controlling for age at onset of signs. We analyzed data on 55 symptomatic patients from the Hunter James Kelly Research Institute's World-Wide Registry. They were tested for galactocerebrosidase activity at Jefferson Medical College (Philadelphia, PA), using survival models in a path model context. Higher galactocerebrosidase activity was predictive of later symptom onset times (P = 0.0011), but did not predict survival after symptom onset (P = 0.9064) when controlling for the logarithm of age at onset. No child with early infantile (aged 0-6 months) phenotype demonstrated galactocerebrosidase activity >0.1 nmol/hour/mg protein. Survival times within a given phenotype did not vary with galactocerebrosidase activity. Although low galactocerebrosidase activity does not predict phenotype, higher activity in the abnormal range (>0.1 nmol/hour/mg protein in this sample) was not identified in the early infantile variant. Galactocerebrosidase activity may be important to consider when predicting phenotype in the newborn screening population. Our findings provide empiric evidence that the upper end (0.15 nmol/hour/mg protein) of the high-risk galactocerebrosidase group in the New York State newborn screening program is conservatively appropriate.

Lysosomal storage disorder 4+1 multiplex assay for newborn screening using tandem mass spectrometry: application to a small-scale population study for five lysosomal storage disorders.

BACKGROUND: We sought to modify a previously published tandem mass spectrometry method of screening for 5 lysosomal storage disorders (LSDs) in order to make it better suited for high-throughput newborn screening. METHODS: Two 3-mm dried blood spot (DBS) punches were incubated, each with a different assay solution. The quadruplex solution was used for screening for Gaucher, Pompe, Krabbe and Fabry diseases, while a separate solution was used for Niemann-Pick A/B disease. RESULTS: The mean activities of acid-ß-glucocerebrosidase (ABG), acid sphingomyelinase (ASM), acid glucosidase (GAA), galactocerebroside-ß-galactosidase (GALC) and acid-galactosidase A (GLA) were measured on 5055 unidentified newborns. The mean activities (compared with their disease controls) were, 15.1 (0.35), 22.2 (1.34), 16.8 (0.51), 3.61 (0.23), and 20.7 (1.43) (µmol/L/h), respectively. The number of specimens that fell below our retest level cutoff of <20% daily mean activity (DMA) for each analyte is: ABG (6), ASM (0), GAA (5), GALC (17), and GLA (2). CONCLUSIONS: This method provides a simplified and reliable assay for screening for five LSDs with clear distinction between activities from normal and disease samples. Advantages of this new method include significant decreases in processing time and the number of required assay solutions and overall decreased complexity.

Clinical signs and neuropathologic abnormalities in working Australian Kelpies with globoid cell leukodystrophy (Krabbe disease).

OBJECTIVE: To characterize the clinical signs of globoid cell leukodystrophy (GLD) in Australian Kelpies from a working line (AWKs) and determine whether an association existed between these signs and degrees of demyelination and inflammatory responses in affected brains. DESIGN: Case-control study. ANIMALS: 4 AWKs with GLD (cases) and 7 unaffected young adult dogs of mixed breeding (controls). PROCEDURES: Clinical records were reviewed for information on signalment, and samples of neurologic tissues underwent histological processing, immunohistochemical staining, and image analysis. Findings were compared between case and control dogs. RESULTS: The 4 affected AWKs had progressive ataxia, tremors, and paresis and low leukocyte activity of galactosylceramidase, the lysosomal enzyme deficient in GLD. Image analysis of neurologic tissue revealed globoid cells characteristic of GLD and substantial demyelination in the peripheral and central nervous systems, relative to that in neurologic tissue from control dogs. This was accompanied by microglial activation, reactive astrocyto-sis, and axonal spheroid formation. CONCLUSIONS AND CLINICAL RELEVANCE: The demyelination, inflammatory responses, and axo-nal spheroids evident in the AWKs were consistent with the clinical signs of peripheral nerve, spinal cord, and cerebellar dysfunction. Because GLD is an autosomal recessive inherited disease, with considerable overlap in galactosylceramidase activity existing among heterozygotes and noncarriers, development of a molecular test is important for preventing the perpetuation of this disease in the Australian Kelpie breed.

Thickening and enhancement of multiple cranial nerves in conjunction with cystic white matter lesions in early infantile Krabbe disease.

We present serial MR findings in a child ultimately diagnosed with the early infantile form of Krabbe disease. MR showed typical features of Krabbe disease including cerebellar and brainstem hyperintensity, periventricular and deep white matter hyperintensity, and cerebral atrophy. In addition, the combination of both enlargement and enhancement of multiple cranial nerves in conjunction with unusual cystic lesions adjacent to the frontal horns of the lateral ventricles was previously unreported and expands the spectrum of imaging findings in early Krabbe disease.

Globoid cell leukodystrophy (Krabbe disease): normal umbilical cord blood galactocerebrosidase activity and polymorphic mutations.

Globoid cell leukodystrophy is an inherited metabolic disorder of the central nervous system caused by deficiency of the lysosomal enzyme galactocerebrosidase. Haematopoietic stem cell transplantation is the only available effective treatment. The engraftment from normal donors provides competent cells able to correct the metabolic defect. Umbilical cord blood cells have proved to significantly decrease complications and improve engraftment rate compared to adult marrow cells in haematopoietic stem cell transplantation. Umbilical cord blood cells must be of sufficient activity to provide central nervous system recovery after engraftment is obtained. Galactocerebrosidase activity is known to be affected by two polymorphic alleles found at nucleotides 502 and 1637 of the cDNA for this gene. This enzyme activity and the polymorphic alleles noted above were analysed in 83 random samples of umbilical cord blood. The activity, assayed with the fluorogenic substrate 6-hexadecanoylamino-4-methylumbelliferyl-beta-galactopyranoside, in those with neither polymorphic allele was 4.6 +/- 1.7 units (nmol/h per mg protein). This optimal choice of cord blood was found in only 24% of specimens. Homozygotes for 1637T > C with activity of only 1.5 +/- 0.4 units represented 16% of the samples. Those heterozygous for 1637T > C with slightly better activity (2.3 +/- 0.7 units) represented 52% of the samples. Choice of umbilical cord blood for haematopoietic stem cell transplantation, therefore, requires consideration not only of cell quantity and HLA compatibility but also selection for normal alleles to obtain maximal enzymatic activity for central nervous system correction.

Measurements from normal umbilical cord blood of four lysosomal enzymatic activities: alpha-L-iduronidase (Hurler), galactocerebrosidase (globoid cell leukodystrophy), arylsulfatase A (metachromatic leukodystrophy), arylsulfatase B (Maroteaux-Lamy).

Umbilical cord blood (UCB) has received increasing attention as a source of unrelated hematopoietic stem cells for transplantation. Lysosomal diseases have been effectively treated and normal enzymatic activity has occurred subsequent to engraftment using UCB. The use of donor cells with normal amounts of enzyme, rather than those from carriers whose level may be 50% or less, is an obvious goal. The frequency of such heterozygotes varies from 1:10 to 1:140 or lower depending upon the disease at issue. We assayed the levels of lysosomal enzymes in normal UCB in random samples as well as those used for transplantation. We measured the following enzymatic activities: alpha-l-iduronidase (Hurler), galactocerebrosidase (globoid cell leuko- dystrophy) and arylsulfatase A (metachromatic leukodystrophy). For the latter, levels of activity in UCB are comparable to those found in adult blood. In the case of arylsulfatase B (Maroteaux-Lamy) a level lower than adult level was found. An informed choice by the transplanting physician based on the activity of the relevant enzyme in the UCB donor will provide a better opportunity for an improved prognosis for more complete correction of the recipient's primary disease. Bone Marrow Transplantation (2000) 25, 541-544.

Late onset globoid cell leukodystrophy (Krabbe's disease)--Swedish case with 15 years of follow-up.

We describe a male patient with late onset globoid cell leukodystrophy (GLD) (Krabbe's disease) still alive at 24 years of age, with a well preserved intellectual and communicative capacity, in contrast to visual failure and severe central pyramidal and extrapyramidal motor disability with spasticity, dystonia, ataxia and peripheral neuropathy. Visual dysfunction began at 4 years of age, limping and balance problems at 8 years and epilepsy at 14 years of age. Neuroimaging at 15 years of age revealed white matter lesions, and nerve conduction velocity examinations showed a slowly developing polyneuropathy. Galactosylceramidase activity was reduced in leukocytes to 0.07 mu kat/kg protein compared with 0.02 (SD 0.01) mu kat/kg protein in infantile GLD.

Arylsulfatase A and beta-galactosidase activities in leukocytes and lymphocytes from normal and psychiatric subjects. Effects of blood-processing delay and interleukin-2 stimulation.

Arylsulfatase A (ASA) and cerebroside-beta-galactosidase activities in leukocytes serve as a diagnostic tool for determining the presence of metachromatic leukodystrophy and globoid cell leukodystrophy, respectively. It has not been demonstrated whether a delay in blood processing and the presence of mixed cell types in different proportions in leukocytes affect the activities of the two enzymes in these cells. We have in the present study determined the specific activity in leukocytes and lymphocytes (T-cells) prepared from blood samples processed immediately after, 4, and 24 h after collection. In order to determine whether the enzyme activities in lymphocytes reflect expression of genetic trait, and not environmental or "state" influence, the activities of the two enzymes in interleukin 2-stimulated T-cells and resting T-cells were compared. A delay of up to 24 h in blood processing did not significantly change the specific activities of the two enzymes in both leukocytes and lymphocytes. The specific activity of ASA and beta-galactosidase in lymphocytes was 1.4-1.8 times that in leukocytes. The activities of the two enzymes in interleukin 2-stimulated T-cells did not differ from those in resting T-cells. These results indicate that blood-processing delay had no significant effects on ASA and beta-galactosidase activity. The data further indicate that the ASA and beta-galactosidase activity in interleukin 2-stimulated T-cells was not significantly different from resting lymphocytes from either normal or psychiatric subjects exposed to various medications. The activity levels in lymphocytes from psychiatric subjects thus reflect expression of genetic trait, rather than environmental or state influence.

Purification and characterization of galactocerebrosidase from human lymphocytes.

Galactocerebrosidase was purified about 22,600-fold using several hydrophobic column and gel filtration steps with a 4.8% recovery, from human lymphocytes. Its specific activity was 1.54 x 10(5) nmol/h/mg with tritium-labeled galactocerebroside as the substrate in the taurocholate system. The optimal pH for galactocerebroside was 4.2 in the taurocholate system and 4.6 in the cholate system. The Km values for galactocerebroside were 5 microM in the taurocholate system and 25 microM in the cholate system. The molecular weight of the purified enzyme was estimated to be 90 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and gel filtration. However, 70, 50, 40, and 30 kDa bands were also recognized on SDS-PAGE. The N-terminal amino acid sequences of the 70 kDa molecule and the three 50 kDa molecules were the same as that of the 90 kDa molecule. The N-terminal amino acid sequences of the 40 and 30 kDa molecules were unique. A monoclonal antibody raised against the purified enzyme effectively immunoprecipitated galactocerebrosidase activity, and an affinity column prepared with this monoclonal antibody bound the 90 and 50 kDa proteins. These results suggest that this enzyme is probably processed from the 90 kDa protein.

Thalamic and basal ganglia hyperdensities--a CT marker for globoid cell leukodystrophy?

Globoid cell leukodystrophy (Krabbe's disease) is a rare autosomal recessive lipidosis, with signs restricted to the nervous system, and is caused by deficiency of the lysosomal hydrolase galactocerebroside beta-galactosidase (galactocerebrosidase). In recent years there have been reports of neurological variants, where age of onset and manifestations differed from the classical form. In other cases, specific CT findings, mainly hyperdensities, have been seen. We report a case of late-infantile Krabbe's disease where thalamic and basal ganglia hyperdensities have been seen on CT scan. We also stress that clinicians should be aware of the possibility of Krabbe's disease in infants who display progressive encephalopathy and these CT findings.

[Comparison of spectrophotometric and fluorimetric methods of determination of galactocerebroside-beta-D-galactosidase in leukocytes]. / Sravnenie spektrofotometricheskogo i fluorimetricheskogo metodov opredeleniia aktivnosti galaktotserebrozid-beta-D-galaktozidazy v leikotsitakh.

A procedure was developed for estimation of galactocerebroside beta-D-galactosidase activity in leukocytes using a new fluorogenic compound 6-hexadecanoylamino-4-methylum-belliferyl-beta-D-galactop yra noside (HMGal) as a substrate. Some patterns of the fluorometric procedure were compared with corresponding parameters of the spectrophotometric method in which a chromogenic substrate HNGal was used. Sensitivity of the fluorometric procedure with HMGal as a substrate was increased 100-fold as compared with the spectrophotometric method. At the same time, the fluorometric procedure enabled to reduce considerably the incubation period and the cell protein content per an assay. High sensitivity and reproducibility of the procedure with HMGal as a substrate allowed to carry out biochemical diagnosis of Krabbe disease in leukocytes.