Galactocerebrosidase Enzyme Activity Research Articles

Improved Brain Pathology and Progressive Peripheral Neuropathy in a 15 Year Old Survivor of Infantile Krabbe Disease Treated With Umbilical Cord Transplantation.

ObjectiveKrabbe disease is a fatal leukodystrophy caused by deficiency in galactocerebrosidase enzyme activity. The only currently available therapy is hematopoietic stem cell transplantation with bone marrow or umbilical cord blood (UCBT), which leads to increased lifespan and functional abilities when performed in the preclinical stage. While stabilization of white matter disease has been seen on serial MRI studies, neuropathological changes following transplantation have not been documented so far.Materials and MethodsWe report the first postmortem examination of a 15-year-old female patient with infantile Krabbe disease after UCBT in infancy.ResultsIn contrast to an untreated Krabbe disease brain, which showed severe myelin and oligodendrocyte loss with occasional globoid cells, the transplanted brain displayed markedly improved myelin preservation, but not reaching normal myelination levels. Consistent with the transplanted patient’s clinical presentation of pronounced deficits in gross motor skills, corticospinal tracts were most severely affected. No globoid cells or evidence of active demyelination were observed in the central nervous system, indicative of at least partially successful functional restoration. This was corroborated by the identification of male donor-derived cells in the brain by in situ hybridization. Unlike the observed disease stabilization in the central nervous system, the patient experienced progressive peripheral neuropathy. While diminished macrophage infiltration was seen postmortem, peripheral nerves exhibited edema, myelin and axon loss and persistent Schwann cell ultrastructural inclusions.ConclusionUmbilical cord blood transplantation was able to alter the natural disease progression in the central but less so in the peripheral nervous system, possibly due to limited cross-correction of Schwann cells.

Fluorimetric assay with a novel substrate for quantification of galactocerebrosidase activity in dried blood spot specimens

BackgroundDecreased galactocerebrosidase (GALC) enzyme activity is causative for Krabbe disease, a lysosomal storage disorder with devastating neurodegenerative consequences. Quantitative fluorimetric assays for GALC activity in isolated blood and skin cells have been described; however, no such assay has been described using dried blood spot (DBS) specimens. MethodsGALC enzyme activity was measured quantitatively using fluorescence from a novel glycosidic substrate: carboxy derived from 6-hexadecanoylamino-4-methylumbelliferone. GALC activity was demonstrated on newborn DBS specimens, known Krabbe disease patient specimens, proficiency testing and quality control samples. ResultsWe present data on characterization of the novel substrate and assay, including pH optimization and enzyme kinetics using a fluorimetric profile. Single and multi-day precision analyses revealed tight analytical measurements with %CV ranging from 5.2% to 14.1%. GALC enzyme activity was linear over the range of 0.31 - 12.04 μmol/l/h with a limit of detection of 0.066 μmol/l/h. Our results with this assay show a clear discrimination between GALC activities in samples from Krabbe disease patients versus presumed normal newborn samples. ConclusionsA fluorimetric assay for GALC enzyme activity measurement on dried blood spot specimens is feasible. Improvements to the assay including novel substrate design, increased substrate concentration and removal of sodium chloride maximize the specificity of the assay and minimize interference from β-galactosidase.

Rare Saposin A deficiency: Novel variant and psychosine analysis

Saposin A is a post-translation product of the prosaposin (PSAP) gene that serves as an activator protein of the galactocerebrosidase (GALC) enzyme, and is necessary for the degradation of certain glycosphingolipids. Deficiency of saposin A leads to a clinical picture identical to that of early-infantile Krabbe disease caused by GALC enzyme deficiency. Galactosylsphingosine, also known as psychosine, is a substrate of the GALC enzyme that is known to be elevated in classic Krabbe disease. We present the case of an 18-month-old male with clinical and radiological findings concerning for Krabbe disease who had preserved GALC enzyme activity and negative GALC gene sequencing, but was found to have a homozygous variant, c.257 T > A (p.I86N), in the saposin A peptide of PSAP. Psychosine determination on dried blood spot at 18 months of age was elevated to 12 nmol/L (normal <3 nmol/L). We present this case to add to the literature on the rare diagnosis of atypical Krabbe disease due to saposin A deficiency, to report a novel presumed pathogenic variant within PSAP, and to suggest that individuals with saposin A deficiency may have elevated levels of psychosine, similar to children with classic Krabbe disease due to GALC deficiency.

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

PurposeNewborn 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). MethodsWe 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. ResultsKrabbe 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. ConclusionBivariate analysis of psychosine and GalC in newborn blood spots can accurately predict early Krabbe symptoms, control false positive rates, and permit presymptomatic treatment.

Adult-onset Krabbe disease in two generations of a Chinese family.

Krabbe disease (KD) is a rare autosomal recessive lysosomal storage disorder caused by deficiency of the galactocerebrosidase (GALC) enzyme. The adult-onset KD is infrequent, and often presenting with slowly progressive spastic paraplegia. Herein, we describe a two-generation concomitant Chinese pedigree of adult-onset KD in which the proband presented with acute hemiplegia at onset. We collected the clinical and neuroimaging data of the pedigree. GALC enzyme activity detection and gene analysis were performed to confirm the diagnosis. Moreover, we reviewed all studies available on PubMed to understand the correlationship between phenotype and genotype of the identified mutations. The proband presented with sudden-onset weakness of left limbs with selective pyramidal tract involvement on diffusion-weighted imaging (DWI) of brain MRI. The GALC enzyme activity of him was low, and the GALC gene analysis revealed compound heterozygous pathogenic mutations of c.1901T>C and c.1901delT. More interestingly, the homozygous c.1901T>C mutations were found in the proband's asymptomatic father and two paternal uncles. Meanwhile, the literature review revealed the c.1901T>C mutation was only found in the late-onset form of KD. These observations, combined with previous reports, indicate that KD should be considered in the adult patients presenting selective pyramidal tract impairment even with sudden onset.

Newborn screening for Krabbe disease in New York State: the first eight years' experience.

Krabbe disease (KD) results from galactocerebrosidase (GALC) deficiency. Infantile KD symptoms include irritability, progressive stiffness, developmental delay, and death. The only potential treatment is hematopoietic stem cell transplantation. New York State (NYS) implemented newborn screening for KD in 2006. Dried blood spots from newborns were assayed for GALC enzyme activity using mass spectrometry, followed by molecular analysis for those with low activity (≤12% of the daily mean). Infants with low enzyme activity and one or more mutations were referred for follow-up diagnostic testing and neurological examination. Of >1.9 million screened, 620 infants were subjected to molecular analysis and 348 were referred for diagnostic testing. Five had enzyme activities and mutations consistent with infantile KD and manifested clinical/neurodiagnostic abnormalities. Four underwent transplantation, two are surviving with moderate to severe handicaps, and two died from transplant-related complications. The significance of many sequence variants identified is unknown. Forty-six asymptomatic infants were found to be at moderate to high risk for disease. The positive predictive value of KD screening in NYS is 1.4% (5/346) considering confirmed infantile cases. The incidence of infantile KD in NYS is approximately 1 in 394,000, but it may be higher for later-onset forms.

A novel homozygous GALC mutation: Very early onset and rapidly progressive Krabbe disease

A clear cut genotype–phenotype correlation for Krabbe disease is not available. Therefore, it is important to identify new mutations and their associated phenotypes to predict the prognosis of the disease. The aim of this study is to identify the causative mutation(s) in a family with Krabbe disease. After a clinical evaluation and suspicion of Krabbe disease galactocerebrosidase activity was analyzed and GALC gene mutation analysis was performed. The galactocerebrosidase enzyme activity was 0.01nmol/mg/h protein (normal range 0.8–4). For further investigation mutation screening was performed by Sanger sequencing across the 17 exons of GALC gene. A novel homozygous mutation c.727delT (p.S243QfsX7) was found. In this study we present the clinical findings along with a novel GALC mutation in a consanguineous Turkish family. Although the relationship between the various genotypes and phenotypes in Krabbe disease has not been fully elucidated an accurate genetic family study is helpful for genetic counseling follow-up and therapy of Krabbe disease. Also, it is important to identify new mutations in order to clarify their clinical importance, to assess the prognosis of the disease, and to suggest either prenatal diagnosis or preimplantation genetic diagnosis to the effected families.