Galactocerebrosidase Research Articles

KRABBE LEUCODYSTROPHY (WITH A DESCRIPTION OF THE CLINICAL CASE)

Krabbe disease (globoid cell leukodystrophy) is a rare autosomal recessive pathology related to lysosomal accumulation diseases. The basis of the disease are mutations in a gene, which encodes the synthesis of the galactocerebrosidase (GALC), which lead to its deficiency. The result is an accumulation with toxic properties predecessor galactocerebroside galactocerebroside (psychosine), which causes the breakdown of myelin with the formation of globoid cells. In 90% of cases, the disease manifests itself within the first six months of life with hyperexcitability, delay or regression of psychomotor development, spasticity of limbs, axial hypotension, lack of reflexes, optic nerve atrophy and microcephaly. In the future, join cramps, neurological status of the patient quickly worsens to decerebration and death before two years of age. Late forms of the disease are characterized by slower progression with much longer life expectancy. The diagnosis of Krabbe disease is facilitated by the results of brain neurovisualization, neurophysiological and histological studies. The final diagnosis is based on the detection of a decrease in GALC activity in blood leukocytes or cultured skin fibroblasts (below 5% of normal), as well as molecular genetic testing, detecting the desired mutation. Neonatal screening often gives a false positive result. Currently, the active search for available biomarkers of the disease for accurate and early diagnosis of the disease is continuing. There are no truly effective treatments for Krabbe disease. Stem cell transplantation is effective only in the asymptomatic stage of the disease, requires myelosuppressive therapy and is associated with the risk of complications. If you have symptoms of a possible supportive therapy, aimed at reducing the excitability and spasticity. Search of substances-activators of residual enzymatic activity GALC is conducted.

Lipid-Conjugated Rigidochromic Probe Discloses Membrane Alteration in Model Cells of Krabbe Disease

The plasma membrane of cells has a complex architecture based on the bidimensional liquid-crystalline bilayer arrangement of phospho- and sphingolipids, which in turn embeds several proteins and is connected to the cytoskeleton. Several studies highlight the spatial membrane organization into more ordered (Lo or lipid raft) and more disordered (Ld) domains. We here report on a fluorescent analog of the green fluorescent protein chromophore that, when conjugated to a phospholipid, enables the quantification of the Lo and Ld domains in living cells on account of its large fluorescence lifetime variation in the two phases. The domain composition is straightforwardly obtained by the phasor approach to confocal fluorescence lifetime imaging, a graphical method that does not require global fitting of the fluorescence decay in every spatial position of the sample. Our imaging strategy was applied to recover the domain composition in human oligodendrocytes at rest and under treatment with galactosylsphingosine (psychosine). Exogenous psychosine administration recapitulates many of the molecular fingerprints of a severe neurological disease, globoid cell leukodystrophy, better known as Krabbe disease. We found out that psychosine progressively destabilizes plasma membrane, as witnessed by a shrinking of the Lo fraction. The unchanged levels of galactosyl ceramidase, i.e., the enzyme lacking in Krabbe disease, upon psychosine treatment suggest that psychosine alters the plasma membrane structure by direct physical effect, as also recently demonstrated in model membranes.

Developmental defects and aberrant accumulation of endogenous psychosine in oligodendrocytes in a murine model of Krabbe disease

Krabbe disease (KD), or globoid cell leukodystrophy, is an inherited lysosomal storage disease with leukodystrophy caused by a mutation in the galactosylceramidase (GALC) gene. The majority of patients show the early onset form of KD dominated by cerebral demyelination with apoptotic oligodendrocyte (OL) death. However, the initial pathophysiological changes in developing OLs remain poorly understood. Here, we show that OLs of twitcher mice, an authentic mouse model of KD, exhibited developmental defects and impaired myelin formation in vivo and in vitro. In twitcher mouse brain, abnormal myelination and reduced expression of myelin genes during the period of most active OL differentiation and myelination preceded subsequent progressive OL death and demyelination. Importantly, twitcher mouse OL precursor cells proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with aberrant accumulation of endogenous psychosine (galactosylsphingosine) and reduced activation of the Erk1/2 and Akt/mTOR pathways before apoptotic cell death. Collectively, our results demonstrate that GALC deficiency in developing KD OLs profoundly affects their differentiation and maturation, indicating the critical contribution of OL dysfunction to KD pathogenesis.

\u03b1-Synuclein interacts directly but reversibly with psychosine: implications for \u03b1-synucleinopathies

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson’s disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe’s disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.

Altered gene transcription linked to astrocytes and oligodendrocytes in frontal cortex in Creutzfeldt-Jakob disease

ABSTRACTTargeted expression of genes coding for proteins specific to astrocytes, oligodendrocytes and myelin was performed in frontal cortex area 8 of Creutzfeldt-Jakob disease methionine/methionine and valine/valine (CJD MM1 and VV2, respectively) compared with controls. GFAP (glial fibrillary acidic protein) mRNA was up-regulated whereas SLC1A2 (solute carrier family 1 member 2, coding for glutamate transporter 1: GLT1), AQ4 (aquaporin 4), MPC1 (mitochondrial pyruvate carrier 1) and UCP5 (mitochondrial uncoupled protein 5) mRNAs were significantly down-regulated in CJD MM1 and CJD VV2, and GJA1 (connexin 43) in CJD VV2. OLIG1 and OLIG2 (oligodendocyte transcription factor 1 and 2, respectively), SOX10 (SRY-Box10) and oligodendroglial precursor cell (OPC) marker NG2 (neuronal/glial antigen) 2 were preserved, but GALC (coding for galactosylceramidase), SLC2A1 (solute carrier family 2 member 1: glucose transporter member 1: GLUT1) and MCT1 (monocarboxylic acid transporter 1) mRNA expression levels were significantly reduced in CJD MM1 and CJD VV2. Expression levels of most genes linked to myelin were not altered in the cerebral cortex in CJD. Immunohistochemistry to selected proteins disclosed individual variations but GFAP, Olig-2, AQ4 and GLUT1 correlated with mRNA levels, whereas GLT1 was subjected to individual variations. However, MPC1, UCP5 and MCT1 decrease was more closely related to the respective reduced neuronal immunostaining. These observations support the idea that molecular deficits linked to energy metabolism and solute transport in astrocytes and oligodendrocytes, in addition to neurons, are relevant in the pathogenesis of cortical lesions in CJD.

A case of late‐onset Krabbe disease which showed subacute progression of spastic paresis with bilateral spinal cord lesions

AbstractWe report a 16‐year‐old boy with the late‐onset form of Krabbe disease. The patient had been free of neuromuscular diseases until he showed subacute progression of spastic paraparesis at age 15. There was no evident peripheral neuropathy. Magnetic resonance imaging (MRI) of the brain and the spinal cord showed T2‐weighted high‐intensity lesions along the bilateral pyramidal tract from the internal capsule to the lumbar cord. Bilateral pyramidal tract involvement raised the possibility of late‐onset Krabbe disease. A markedly reduced galactocerebrosidase activity and a homozygous known pathogenic mutation in the GALC gene confirmed the diagnosis. This case was unique in that the progression was subacute and the lesions from the cervical to the lumbar cord were clearly shown by MRI.

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.

Unfolded protein response is activated in Krabbe disease in a manner dependent on the mutation type.

Krabbe disease, one of the autosomal-recessive lysosomal storage disorders (LSDs), is caused by a deficiency of galactocerebrosidase (GALC) activity, resulting in the intracellular accumulation of psychosine, which is cytotoxic for neuronal cells. Genetically pathogenic mutations result in conformational changes in GALC and disrupt the lysosmal trafficking of cargos, which subsequently accumulate in the endoplasmic reticulum (ER). Recently, ER stress together with the activation of the unfolded protein response (UPR) has been suggested to play a key role in the pathogenesis of LSDs. In this study, we hence investigated whether the UPR is activated in Krabbe disease using COS-7 cells expressing pathogenic GALC mutants and skin fibroblasts (SFs) from Krabbe disease patients with various phenotypes, using a combination of semiquantitative and quantitative real-time polymerase chain reactions. We found that UPR activation in Krabbe disease depends on the mutations and cell types, and there is the possibility that multiple pathways, involving ER chaperones, inositol-requiring kinase 1, and protein kinase regulated by RNA-like ER kinase are activated by mutations associated with the infantile form. These results indicate that in Krabbe disease, each misfolded/unfolded protein evokes different UPR activation depending on the mutation, and that the activated pathways affect the phenotypes.

Long-Term Improvement of Neurological Signs and Metabolic Dysfunction in a Mouse Model of Krabbe's Disease after Global Gene Therapy.

We report a global adeno-associated virus (AAV)9-based gene therapy protocol to deliver therapeutic galactosylceramidase (GALC), a lysosomal enzyme that is deficient in Krabbe's disease. When globally administered via intrathecal, intracranial, and intravenous injections to newborn mice affected with GALC deficiency (twitcher mice), this approach largely surpassed prior published benchmarks of survival and metabolic correction, showing long-term protection of demyelination, neuroinflammation, and motor function. Bone marrow transplantation, performed in this protocol without immunosuppressive preconditioning, added minimal benefits to the AAV9 gene therapy. Contrasting with other proposed pre-clinical therapies, these results demonstrate that achieving nearly complete correction of GALC's metabolic deficiencies across the entire nervous system via gene therapy can have a significant improvement to behavioral deficits, pathophysiological changes, and survival. These results are an important consideration for determining the safest and most effective manner for adapting gene therapy to treat this leukodystrophy in the clinic.

Heterozygous carriers of galactocerebrosidase mutations that cause Krabbe disease have impaired microglial function and defective repair of myelin damage.

This review addresses two puzzling findings related to mutations in galactocerebrosidase (GALC) that cause Krabbe disease (KD), a severe lysosomal storage disorder characterized by extensive myelin damage in children with mutations in both GALC alleles. First, heterozygous carriers of KD-causing mutations, which include the biological parents of children with KD, exhibit increased risk for developing other diseases. Second, variants in the GALC locus increase the risk of developing multiple sclerosis (MS), another disease characterized by extensive myelin damage. What explains these correlations? In studies on cuprizone-induced myelin damage in heterozygous (GALC+/–) mice carrying one copy of a mutation that causes KD-like disease, the extent of damage was similar in GALC+/– and wild-type (WT) mice. In contrast, GALC+/- mice had striking defects in repair of cuprizone-induced damage. We further found unexpected microglial defects in myelin debris clearance and in the ability to up-regulate the Trem2 microglial protein critical for debris uptake. These defects were rescued by exposure to a lysosomal re-acidifying drug discovered in our studies on KD, and which provides multiple clinically relevant benefits in the twitcher (GALC+/–) mouse model of KD. Thus, heterozygous GALC mutations cause effects on biological function that may help to understand the increased disease risk in heterozygous carriers of such mutations and to understand why GALC variations increase the risk of MS. Our findings indicate that while some genetic risk factors may contribute to complex diseases by increasing the risk of tissue damage, others may do so by compromising tissue repair.

Phospholipase A2 is involved in galactosylsphingosine-induced astrocyte toxicity, neuronal damage and demyelination.

Krabbe disease is a fatal rare inherited lipid storage disorder affecting 1:100,000 births. This illness is caused by mutations in the galc gene encoding for the enzyme galactosylceramidase (GALC). Dysfunction of GALC has been linked to the toxic build-up of the galactolipid, galactosylsphingosine (psychosine), which induces cell death of oligodendrocytes. Previous studies show that phospholipase A2 (PLA2) may play a role in psychosine induce cell death. Here, we demonstrate that non-selective inhibition of cPLA2/sPLA2 and selective inhibition of cPLA2, but not sPLA2, also attenuates psychosine-induced cell death of human astrocytes. This study shows that extracellular calcium is required for psychosine induced cell death, but intracellular calcium release, reactive oxygen species or release of soluble factors are not involved. These findings suggest a cell autonomous effect, at least in human astrocytes. Supporting a role for PLA2 in psychosine-induced cell death of oligodendrocytes and astrocytes, the results show inhibition of PLA2 attenuates psychosine-induced decrease in the expression of astrocyte marker vimentin as well as myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and the neuronal marker SMI-32 in organotypic slice cultures. These findings provide further mechanistic details of psychosine-induced death of glia and suggest a role for PLA2 in the process. This work also supports the proposal that novel drugs for Krabbe disease may require testing on astrocytes as well as oligodendrocytes for more holistic prediction of pre-clinical and clinical efficacy.

Importance of phenotype estimation for Krabbe disease with a newly developed diagnostic procedure in the era of advanced medical treatment

Krabbe disease is one of the metabolic leukodystrophies caused by dysfunction of the hydrophobic lysosomal enzyme, galactocerebrosidase (EC 3.2.1.46) which is encoded by the GALC gene. Krabbe disease was first reported as a clinical entity 100 years ago, in 1916, by Knud Krabbe (1). The disease frequency is about 1/100,000–1/200,000 (2). It is recognized as one of the predominant genetic diseases, showing leukodystrophy from infancy to adulthood.

Heterozygote galactocerebrosidase (GALC) mutants have reduced remyelination and impaired myelin debris clearance following demyelinating injury.

Genome-wide association studies are identifying multiple genetic risk factors for several diseases, but the functional role of these changes remains mostly unknown. Variants in the galactocerebrosidase (GALC) gene, for example, were identified as a risk factor for Multiple Sclerosis (MS); however, the potential biological relevance of GALC variants to MS remains elusive. We found that heterozygote GALC mutant mice have reduced myelin debris clearance and diminished remyelination after a demyelinating insult. We found no histological or behavioral differences between adult wild-type and GALC +/- animals under normal conditions. Following exposure to the demyelinating agent cuprizone, however, GALC +/- animals had significantly reduced remyelination during recovery. In addition, the microglial phagocytic response and elevation of Trem2, both necessary for clearing damaged myelin, were markedly reduced in GALC +/- animals. These altered responses could be corrected in vitro by treatment with NKH-477, a compound discovered as protective in our previous studies on Krabbe disease, which is caused by mutations in both GALC alleles. Our data are the first to show remyelination defects in individuals with a single mutant GALC allele, suggesting such carriers may have increased vulnerability to myelin damage following injury or disease due to inefficient myelin debris clearance. We thus provide a potential functional link between GALC variants and increased MS susceptibility, particularly due to the failure of remyelination associated with progressive MS. Finally, this work demonstrates that genetic variants identified through genome-wide association studies may contribute significantly to complex diseases, not by driving initial symptoms, but by altering repair mechanisms.

Psychosine, a marker of Krabbe phenotype and treatment effect.

Newborn screening (NBS) for Krabbe disease, a rare neurodegenerative disorder caused by deficient galactocerebrosidase (GALC) enzyme activity, has recently been implemented in a number of US states. However, the spectrum of phenotypic manifestations associated with deficient GALC activity complicates the management of screen-positive newborns and underscores the need to identify clinically relevant biomarkers. Earlier studies with a small number of patients identified psychosine, a substrate of the GALC enzyme, as a potential biomarker for Krabbe disease. In this study, we provide, for the first time, longitudinal data on dried blood spot (DBS) psychosine concentrations in different Krabbe disease phenotypes for both untreated patients and those treated with hematopoietic stem cell transplantation (HSCT). Our cohort included patients previously identified by NBS to be at high risk to develop Krabbe disease. Substantially elevated DBS psychosine concentration during the newborn period was found to be a highly specific marker for infantile Krabbe disease. This finding supports the use of DBS psychosine concentration as a second-tier NBS test to aid in the identification of patients who require urgent evaluation for HSCT. In addition, longitudinal assessments showed that both natural disease progression and treatment with HSCT were associated with decreases in DBS psychosine concentrations. Based on these findings we provide recommendations for the interpretation of psychosine concentrations in DBS specimens collected during the first year of life. Future studies should aim to better delineate the relationship between DBS psychosine concentration and disease onset in patients with later-onset forms of Krabbe disease.

A Specific Activity‐Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease.

Detecting multiple lysosomal enzymes in dried blood spots by tandem mass spectrometry

Objective Establish a method for measuring the activities of Galactocerebrosidase (GALC), α-Glucosidase(GAA), α-Galactosidase (GLA) and α-L-Iduronidase (IDUA) in dried blood spots specimen by tandem mass spectrometry (MS/MS). Methods A total of 2175 dried blood spot samples forinborn errors of metabolism in neonatalscreening center of Shanghai Xinhua hospital were collected in July and November, 2013.And twenty dried blood spot samples from patients withlysosomalstorage disorders(LSDs)of Shanghai Xin Hua Hospital were collected from September 2012 to January 2014. The extraction of DBS was incubated with enzyme substrates and internal standards.After liquid-liquid and solid-phase extraction, the extraction solution was dried under nitrogen and reconstituted. Then enzyme reaction products and internal standards were analyzed by MS/MS. Linearity, precision, accuracy and the limit of detection were evaluated. 2175 dried blood spot samples were detected to establish the normal reference range for the activities of four enzymes according to 0.5th to 99.5th percentiles. 20 specimens from patients withLSDs were detected to verify the reference range inclinical judgment. Results The intraassay and interassay precisions ranged from 1.7% to 11.8%, and the intraassay and interassay accuracies ranged from 85% to 115%. The linear coefficients for measured concentration of enzyme products/internal standards and theoretical concentration were 0.997-0.999. The limits of detection forGALC, GAA, GLA and GLA were 0.03 μmol/(L·h), 0.09 μmol/(L·h), 0.12 μmol/(L·h) and 0.16 μmol/(L·h). The normal reference values for GALC, GAA, GLA and GLAwere 0.51-8.51 μmol(L·h), 1.99-22.22 μmol/(L·h), 1.68-41.59 μmol/(L·h) and 2.36-19.21 μmol/(L·h). The enzymes of 20 patients with LSDs were remarkably decreased compared to the normal range. The Krabbe, Pompe, Fabry, MPSⅠ patients can be effectively detected by this MS/MS method. Conclusions A MS/MS method for measuring GALC, GAA, GLA and IDUA enzyme activities in DBShas been established.(Chin J Lab Med, 2016, 39: 761-765) Key words: Lysosomal storage diseases; Tandem mass spectrometry; Dried blood spot testing; Galactosylceramidase; Alpha-Glucosidases; Alpha-Galactosidase; Iduronidase

Glycosynthase mediated synthesis of psychosine

Globoid cell leukodystrophy (GCL), or Krabbe disease, is a lysosomal storage disorder characterized by a deficiency in galactosylceramidase (GALC), which hydrolyses galactosylceramide and galactosylsphingosine (psychosine). Early detection of GCL in newborns is essential for timely therapeutic intervention and could be achieved by testing infant blood samples with isotopically labeled lysosmal enzyme substrates and mass spectrometry. While isotopically labeled psychosine would be a useful tool for the early diagnosis of GCL, its synthesis is lengthy and expensive. To obviate this problem we developed a one-step chemoenzymatic synthesis of psychosine using a glycosynthase mutant of the Rhodococcus equi endogalactosylceramidase (EGALC), α-D-galactopyranosyl fluoride and sphingosine.

Expression of individual mutations and haplotypes in the galactocerebrosidase gene identified by the newborn screening program in New York State and in confirmed cases of Krabbe's disease.

Newborn screening (NBS) for Krabbe's disease (KD) has been instituted in several states, and New York State has had the longest experience. After an initial screening of dried blood spots, samples from individuals with galactocerebrosidase (GALC) values below a given cutoff level were subjected to additional testing, including sequencing of the GALC gene. This resulted in the identification of mutations that had previously been found in confirmed KD patients and of variants that had never previously been reported. Some individuals had variants considered to be polymorphisms, alone or on the same allele as another mutation. To help with counseling of families on the risk for a newborn to develop KD, expression studies were conducted with these variants identified by NBS. GALC activity was measured in COS1 cells for 140 constructs and compared with mutations that had previously been seen in confirmed cases of KD. When a polymorphism was present on the same allele as the variant, expressed activity was measured with and without the polymorphism. In some cases the presence of the polymorphism greatly lowered the measured GALC activity, possibly making it disease causing. Although it is not possible to predict conclusively whether a variant is severe and will result in infantile KD if two such variants are present or whether a variant is mild and will result in late-onset disease, some variants clearly are not disease causing. This is the largest expression study of GALC variants/mutations found in NBS and confirmed KD cases. This work will be helpful for counseling families of screen-positive newborns found to have low GALC activity. © 2016 Wiley Periodicals, Inc.

Can psychosine and galactocerebrosidase activity predict early-infantile Krabbe's disease presymptomatically?

Krabbe's disease (KD) is a fatal neurodegenerative disorder, with the early-infantile form (EIKD) defined by onset of symptoms before age 6 months. Early and highly accurate identification of EIKD is required to maximize benefits of hematopoietic stem cell transplantation treatment. This study investigates the potential for accurate prediction of EIKD based on a novel newborn screening (NBS) tool developed from two biomarkers, galactocerebrosidase (GALC) enzyme activity and galactosylsphingosine concentration (psychosine [PSY]). Normative information about PSY and GALC, derived from distinct samples of normal newborns, was used to develop the novel diagnostic tool. Bivariate normal limits (BVNL) were constructed, assuming a multivariate normal distribution of natural logarithms of GALC and PSY of normal newborns. The (lnGALC, lnPSY) points for newborns in various "abnormal groups," including one group of infants who subsequently suffered EIKD, were plotted on a graph of BVNL. The points for all EIKD patients fell outside of BVNL (100% sensitivity). In a simulation study to compare the false-positive rate of existing univariate methods of diagnosis with our new BVNL-based method, we generated 100 million normal newborn data points. All fell within BVNL (i.e., zero false positives), whereas 5,682 false positives were observed when applying a two-tiered univariate method of the type suggested in the literature. These results suggest that (lnGALC, lnPSY) BVNLs will allow highly accurate prediction of EIKD, whereas two-tiered univariate approaches will not. Redevelopment of the BVNL based on GALCs and PSYs measured on a common large sample of normal newborns is required for NBS use. © 2016 Wiley Periodicals, Inc.

Treatment for Krabbe's disease: Finding the combination.

Globoid cell leukodystrophy (GLD) is an autosomal recessive neurodegenerative disorder caused by a deficiency of the lysosomal enzyme galactocerebrosidase (GALC). GALC is responsible for catabolism of certain glycolipids, including the toxic compound galactosylsphingosine (psychosine). Histological signs of disease include the widespread loss of myelin in the central and peripheral nervous systems, profound neruroinflammation, and axonal degeneration. Patients suffering from GLD also display neurological deterioration. Many different individual therapies have been investigated in the murine model of the GLD, the Twitcher mouse, with minimal success. The current standard of care for GLD patients, hematopoietic stem cell transplantation, serves only to delay disease progression and is not an effective cure. However, combination therapies that target different pathogenic mechanisms/pathways have been more effective at reducing histological signs of disease, delaying disease onset, prolonging life span, and improving behavioral/cognitive functions in rodent models of Krabbe's disease. In some cases, dramatic synergy between the various therapies has been observed. © 2016 Wiley Periodicals, Inc.