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Objective: To provide updated evidence and consensus-based recommendations for the classification of individuals who screen positive for Krabbe Disease (KD) and recommendations for long-term follow-up for those who are at risk for late onset Krabbe Disease (LOKD)., Methods: KD experts (KD NBS Council) met between July 2017 and June 2020 to develop consensus-based classification and follow-up recommendations. The resulting newly proposed recommendations were assessed in a historical cohort of 47 newborns from New York State who were originally classified at moderate or high risk for LOKD., Results: Infants identified by newborn screening with possible KD should enter one of three clinical follow-up pathways (Early infantile KD, at-risk for LOKD, or unaffected), based on galactocerebrosidase (GALC) activity, psychosine concentration, and GALC genotype. Patients considered at-risk for LOKD based on low GALC activity and an intermediate psychosine concentration are further split into a high-risk or low-risk follow-up pathway based on genotype. Review of the historical New York State cohort found that the updated follow-up recommendations would reduce follow up testing by 88%., Conclusion: The KD NBS Council has presented updated consensus recommendations for efficient and effective classification and follow-up of NBS positive patients with a focus on long-term follow-up of those at-risk for LOKD., Competing Interests: Declaration of competing interest Robert Thompson Stone, MD: None. Margie A. Ream, MD: A member of the Evidence Review Group for the Advisory Committee on Heritable Disorders in Newborns and Children. The views expressed herein are solely those of the authors and do not necessarily reflect the views of the Advisory Committee on Heritable Disorders in Newborns and Children, or the members of the Evidence Review Group. Michael Gelb, PhD: Consultant for PerkinElmer Inc. Dietrich Matern, MD, PhD: None. Joseph J. Orsini, PhD: None. Paul A. Levy, MD: None. Jennifer P. Rubin, MD: None. David A. Wenger, PhD: None. Barbara K. Burton, MD: Has received consulting fees and/or honoraria from Biomarin, Shire (Takeda), Sanofi Genzyme, Horizon, Alexion, Moderna, Denali, JCR Pharma, Aeglea, Inventiva and Ultragenyx. She has conducted clinical trials funded by Biomarin, Shire (Takeda), Ultragenyx, Sangamo and Homology Medicines. Maria L. Escolar, MD: Advisory Boards Orphazyme, Shire/Takeda. Consulting Fees, Sanofi, AvroBio, JCR. Contracted Research Abeona, Prevail, Denali, Shire/Takeda, Regenxbio. Salary/Ownership Interest greater than 5%, Forge Biologics, Chief Medical Officer. Speaker's Bureau and Travel Expenses Shire/Takeda, Sanofi. Joanne Kurtzberg, MD: None., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Krabbe disease is a rare neurological disorder caused by a deficiency in the lysosomal enzyme, β-galactocerebrosidase, resulting in demyelination of the central and peripheral nervous systems. If left without treatment, Krabbe disease results in progressive neurodegeneration with reduced quality of life and early death. The purpose of this prospective study was to describe the natural progression of early onset Krabbe disease in a large cohort of patients., Methods: Patients with early onset Krabbe disease were prospectively evaluated between 1999 and 2018. Data sources included diagnostic testing, parent questionnaires, standardized multidisciplinary neurodevelopmental assessments, and neuroradiological and neurophysiological tests., Results: We evaluated 88 children with onset between 0 and 5 months. Median age of symptom onset was 4 months; median time to diagnosis after onset was 3 months. The most common initial symptoms were irritability, feeding difficulties, appendicular spasticity, and developmental delay. Other prevalent symptoms included axial hypotonia, abnormal deep tendon reflexes, constipation, abnormal pupillary response, scoliosis, loss of head control, and dysautonomia. Results of nerve conduction studies showed that 100% of patients developed peripheral neuropathy by 6 months of age. Median galactocerebrosidase enzyme activity was 0.05 nmol/h/mg protein. The median survival was 2 years., Conclusions: This is the largest prospective natural history study of Krabbe disease. It provides a comprehensive description of the disease during the first 2 years of life. With recent inclusion of state mandated newborn screening programs and promising therapeutic interventions, enhancing our understanding of disease progression in early onset Krabbe disease will be critical for developing treatments, designing clinical trials, and evaluating outcomes.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is an inherited, neurologic disorder that results from deficiency of a lysosomal enzyme, galactosylceramidase. Most commonly, deficits of galactosylceramidase result in widespread central and peripheral nervous system demyelination and death in affected infants typically by 2 years of age. Hematopoietic stem-cell transplantation is the current standard of care in children diagnosed prior to symptom onset. However, disease correction is incomplete. Herein, the first adeno-associated virus (AAV) gene therapy experiments are presented in a naturally occurring canine model of GLD that closely recapitulates the clinical disease progression, neuropathological alterations, and biochemical abnormalities observed in human patients. Adapted from studies in twitcher mice, GLD dogs were treated by combination intravenous and intracerebroventricular injections of AAVrh10 to target both the peripheral and central nervous systems. Combination of intravenous and intracerebroventricular AAV gene therapy had a clear dose response and resulted in delayed onset of clinical signs, extended life-span, correction of biochemical defects, and attenuation of neuropathology. For the first time, therapeutic effect has been established in the canine model of GLD by targeting both peripheral and central nervous system impairments with potential clinical implications for GLD patients.

Background: Krabbe disease is a rare neurodegenerative genetic disorder caused by deficiency of galactocerebrosidase. Patients with the infantile form of Krabbe disease can be treated at a presymptomatic stage with human stem cell transplantation which improves survival and clinical outcomes. However, without a family history, most cases of infantile Krabbe disease present after onset of symptoms and are ineligible for transplantation. In 2006, New York began screening newborns for Krabbe disease to identify presymptomatic cases. To ensure that those identified with infantile disease received timely treatment, New York public health and medical systems took steps to accurately diagnose and rapidly refer infants for human stem cell transplantation within the first few weeks of life. After 11 years of active screening in New York and the introduction of Krabbe disease newborn screening in other states, new information has been gained which can inform the design of newborn screening programs to improve infantile Krabbe disease outcomes., Findings: Recent information relevant to Krabbe disease screening, diagnosis, and treatment were assessed by a diverse group of public health, medical, and advocacy professionals. Outcomes after newborn screening may improve if treatment for infantile disease is initiated before 30 days of life. Newer laboratory screening and diagnostic tools can improve the speed and specificity of diagnosis and help facilitate this early referral. Given the rarity of Krabbe disease, most recommendations were based on case series or expert opinion., Conclusion: This report updates recommendations for Krabbe disease newborn screening to improve the timeliness of diagnosis and treatment of infantile Krabbe disease. In the United States, several states have begun or are considering Krabbe disease newborn screening. These recommendations can guide public health laboratories on methodologies for screening and inform clinicians about the need to promptly diagnose and treat infantile Krabbe disease. The timing of the initial referral after newborn screening, the speed of diagnostic confirmation of infantile disease, and the transplantation center's experience and ability to rapidly respond to a suspected patient with newly diagnosed infantile Krabbe disease are critical for optimal outcomes.

Background: Early infantile Krabbe disease is rapidly fatal, but hematopoietic stem cell transplantation (HSCT) may improve outcomes if performed soon after birth. New York State began screening all newborns for Krabbe disease in 2006., Methods: Infants with abnormal newborn screen results for Krabbe disease were referred to specialty-care centers. Newborns found to be at high risk for Krabbe disease underwent a neurodiagnostic battery to determine the need for emergent HSCT., Results: Almost 2 million infants were screened. Five infants were diagnosed with early infantile Krabbe disease. Three died, two from HSCT-related complications and one from untreated disease. Two children who received HSCT have moderate to severe developmental delays. Forty-six currently asymptomatic children are considered to be at moderate or high risk for development of later-onset Krabbe disease., Conclusions: These results show significant HSCT-associated morbidity and mortality in early infantile Krabbe disease and raise questions about its efficacy when performed in newborns diagnosed through newborn screening. The unanticipated identification of "at risk" children introduces unique ethical and medicolegal issues. New York's experience raises questions about the risks, benefits, and practicality of screening newborns for Krabbe disease. It is imperative that objective assessments be made on an ongoing basis as additional states begin screening for this disorder.Genet Med 18 12, 1235-1243.

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., (© 2016 Wiley Periodicals, Inc.)

This Review summarizes the progress in understanding the pathogenesis and treatment of Krabbe disease from the description of five patients in by Knud Krabbe until 2016. To determine the cause of this genetic disease, pathological and chemical analyses of tissues from the nervous systems of patients were performed. It was determined that these patients had a pathological feature known as globoid cell in the brain and that this consisted partially of galactosylceramide, a major sphingolipid component of myelin. The finding that these patients had a deficiency of galactocerebrosidase (GALC) activity opened the way to relatively simple diagnostic testing with easily obtainable tissue samples, studies leading to the purification of GALC, and cloning of the GALC cDNA and gene. The availability of the gene sequence led to the identification of mutations in patients and to the current studies involving the use of viral vectors containing the GALC cDNA to treat experimentally naturally occurring animal models, such as twitcher mice. Currently, treatment of presymptomatic human patients is limited to hematopoietic stem cell transplantation (HSCT). With recent studies showing successful treatment of animal models with a combination of HSCT and viral gene therapy, it is hoped that more effective treatments will soon be available for human patients. For this Review, it is not possible to reference all of the articles contributing to our current state of knowledge about this disease; however, we have chosen those that have influenced our studies by suggesting research paths to pursue. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Purpose: 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., Methods: 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., Results: 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., Conclusions: 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.

Krabbe disease is an autosomal recessive disorder resulting from defects in the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency leads to severe neurological features. The only treatment for presymptomatic infantile patients and later-onset patients is hematopoietic stem cell transplantation (HSCT). This treatment is less than ideal with most patients eventually developing problems with gait and expressive language. Several naturally occurring animal models are available, including twitcher (twi) mice, which have been used for many treatment trials. Previous studies demonstrated that multiple injections of AAVrh10-GALC into the central nervous system (CNS) of neonatal twi mice resulted in significant improvements. Recently we showed that one i.v. injection of AAVrh10-GALC on PND10 resulted in normal GALC activity in the CNS and high activity in the peripheral nervous system (PNS). In the present study, a single i.v. injection of AAVrh10-GALC was given 1 day after bone marrow transplantation (BMT) on PND10. The mice show greatly extended lifespan and normal behavior with improved CNS and PNS findings. Since HSCT is the standard of care in human patients, adding this single i.v. injection of viral vector may greatly improve the treatment outcome.

An inherited deficiency of β-galactosylceramidase (GALC) causes the lysosomal storage disease globoid cell leukodystrophy (GLD). The disease is characterized by the accumulation of the cytotoxic metabolite psychosine (galactosylsphingosine), causing rapid degeneration of myelinating cells. Most patients suffer from the infantile form of GLD with onset of disease between 3 and 6 months after birth and death by 2 years of age. The most widely used animal model of GLD, the twitcher mouse, presents with an even more rapid course of disease and death around 40 days of age. We have generated a novel "humanized" mouse model of GLD by inserting a human GALC cDNA containing an adult-onset patient mutation into the murine GALC gene. Humanized GALC mice exhibit pathological hallmarks of GLD including psychosine accumulation, neuroinflammation, CNS infiltration of macrophages, astrogliosis and demyelination. Residual GALC activities in mouse tissues are low and the mice display a median lifespan of 46 days. Due to the expression of the human transgene, the mice do not develop an immune response against rhGALC, rendering the animal model suitable for therapies based on human enzyme. Intravenously injected rhGALC was able to surmount the blood-brain barrier and was targeted to lysosomes of brain macrophages, astrocytes and neurons. High-dose enzyme replacement therapy started at postnatal day 21 reduced the elevated psychosine levels in the peripheral and central nervous system by 14-16%, but did not ameliorate neuroinflammation, demyelination and lifespan. These results may indicate that treatment must be started earlier before pathology occurs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessive disorder resulting from the defective lysosomal enzyme galactocerebrosidase (GALC). The lack of GALC enzyme leads to severe neurological symptoms. While most human patients are infants who do not survive beyond 2 years of age, older patients are also diagnosed. In addition to human patients, several naturally occurring animal models, including dog, mouse, and monkey, have also been identified. The mouse model of Krabbe disease, twitcher (twi) mouse has been used for many treatment trials including gene therapy. Using the combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of the adeno-associated virus serotype rh10 (AAVrh10) expressing mouse GALC in neonate twi mice we previously have demonstrated a significantly extended normal life and exhibition of normal behavior in treated mice. In spite of the prolonged healthy life of these treated mice and improved myelination, it is unlikely that using multiple injection sites for viral administration will be approved for treatment of human patients. In this study, we have explored the outcome of the single iv injection of viral vector at post-natal day 10 (PND10). This has resulted in increased GALC activity in the central nervous system (CNS) and high GALC activity in the peripheral nervous system (PNS). As we have shown previously, an iv injection of AAVrh10 at PND2 results in a small extension of life beyond the typical lifespan of the untreated twi mice (~40 days). In this study, we report that mice receiving a single iv injection at PND10 had no tremor and continued to gain weight until a few weeks before they died. On average, they lived 20-25 days longer than untreated mice. We anticipate that this strategy in combination with other therapeutic options may be beneficial and applicable to treatment of human patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: New York State has screened over 1.2 million newborns for Krabbe disease, and we identified 4 newborns with infantile Krabbe disease. In addition, 6 other newborns were identified with very low galactosylcerebrosidase (GALC) activity. Because these patients remain asymptomatic, we investigated whether psychosine levels could be a useful marker for disease., Methods: HPLC-MS/MS methodology was used to determine the psychosine concentrations in dried blood spots (DBS) collected from the following cohorts: known Krabbe patients, screened babies that were determined to have infantile Krabbe disease, asymptomatic infants with low GALC activity, and normal controls., Results: The psychosine concentrations from the known Krabbe patients ranged from 7 to 50 ng/ml. Newborns identified by screening who were confirmed with infantile Krabbe disease ranged from 23 to 73 ng/ml. Asymptomatic individuals with low GALC activity had concentrations ranging from 1.7 to 5.7 ng/ml. Concentrations in newborns with normal GALC activity were all <3 ng/ml., Conclusions: The psychosine concentrations in DBS from confirmed infantile patients are at least four times higher than the asymptomatic newborns and nearly an order of magnitude greater than normal newborns. Further studies are needed to determine if psychosine can be used as a predictor of disease status/progression in screen positive newborns., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). This deficiency results in accumulation of certain galactolipids including psychosine which is cytotoxic for myelin-producing cells. Treatment of human patients at this time is limited to hematopoietic stem cell transplantation (HSCT) that appears to slow the progression of the disease when performed in presymptomatic patients. In this study, adeno-associated virus (AAV) serotype rh10-(AAVrh10) expressing mouse GALC was used in treating twitcher (twi) mice, the mouse model of GLD. The combination of intracerebroventricular, intracerebellar, and intravenous (iv) injection of viral particles in neonate twi mice resulted in high GALC activity in brain and cerebellum and moderate to high GALC activity in spinal cord, sciatic nerve, and some peripheral organs. Successfully treated mice maintained their weight with no or very little twitching, living up to 8 months. The physical activities of the long-lived treated mice were comparable to wild type for most of their lives. Treated mice showed normal abilities to mate, to deliver pups, to nurse and to care for the newborns. This strategy alone or in combination with other therapeutic options may be applicable to treatment of human patients.

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). GALC deficiency results in a progressive demyelination of the central and peripheral nervous systems. Inflammatory cells and increased levels of cytokines and chemokines are present in the CNS of GLD mice and may play a significant role in the pathogenesis of the disease. In this study we evaluate the effect of non-steroidal anti-inflammatory drugs, such as indomethacin and ibuprofen, and minocycline, a tetracycline analog with neuroprotective and anti-apoptotic properties, on the progression of the disease using a transgenic mouse model of GLD. Real-time quantitative PCR was used to analyze the expression of several markers of the immune/inflammatory response. IL-6, TNF-alpha, MIP-1beta, MCP-1, iNOS/NOS2, CD11b, CD68, CD4 and CD8 mRNA levels were measured in cortex, cerebellum and spinal cord of untreated and treated affected mice at different ages. In addition, the pharmacological treatments were compared to bone marrow transplantation (BMT). The pharmacological treatments significantly extended the life-span of the treated mice and reduced the levels of several of the immuno-related factors studied. However, BMT produced the most dramatic improvements. In BMT-treated mice, factors in the spinal cord were normalized faster than the cerebellum, with the exception of CD68. There was a decrease in the number of apoptotic cells in the cerebellum of mice receiving anti-inflammatory drugs and BMT. These studies indicate a possible role for combined therapy in the treatment of GLD.

Krabbe disease or globoid cell leukodystrophy is an autosomal recessive disorder resulting from mutations in the galactocerebrosidase (GALC) gene. These mutations lead to deficient GALC activity, storage of substrates of the enzyme, including psychosine, death to oligodendrocytes, decreased myelination, production of globoid cells and eventually death to the individual. While most affected individuals are infants, late-onset forms are also recognized. In addition to human patients, several animal models have been well characterized, including the twitcher mouse. A spontaneously transformed progenitor cell line was isolated from an astrocyte-enriched fraction of normal mice, partially characterized and transduced with a retrovirus-containing mouse GALC cDNA to produce increased GALC activity (20-30-fold above baseline). These cells, called MAR-52, were injected into the brains of newborn affected twitcher mice. While there was only a modest increase in lifespan and body weight, there was clear evidence for the correction of the astrocytic gliosis, normal appearing oligodendrocytes and evidence for remyelination. We demonstrate that the exogenously supplied neural progenitor cells can donate GALC enzyme to oligodendrocytes in the brains of affected mice resulting in normal myelination in the area of donor cells. At this time, hematopoietic stem cell transplantation provides the best outcome in affected mice and is the only treatment available for human patients, but it does not result in a cure even when performed in asymptomatic newborns. Complete correction probably will require a combined approach to effectively treat patients with Krabbe disease. With developments in the isolation and characterization of stem cells, this approach may improve the outcome for individuals diagnosed in the future.

Krabbe disease is a rare inherited neurologic disorder affecting the central and peripheral nervous systems. The disease has four phenotypes: early infantile, later onset, adolescent, and adult. The only known treatment is hematopoietic stem cell transplantation, which is, in the early infantile form of the disease, most beneficial if performed before onset of clinical symptoms. In August 2006, New York State began screening all newborns for Krabbe disease. A rapid and accurate technique for assessing galactocerebrosidase activity and performing DNA mutation analysis had been developed. Interpreting these results was limited, however, because neither enzyme activity nor genetic mutation reliably predicts phenotype. A series of initiatives were therefore developed by a multidisciplinary group of neurologists, geneticists, metabolic pediatricians, neurodevelopmental pediatricians, and transplant physicians (the Krabbe Consortium of New York State) to enhance the effectiveness of the newborn screening program. A standardized clinical evaluation protocol was designed based on the available literature, criteria for transplantation for the early infantile phenotype were formulated, a clinical database and registry was developed, and a study of developmental and functional outcomes was instituted. This multidisciplinary standardized approach to evaluating infants who have positive results on newborn screening may serve as a model for other states as they begin the process of screening for Krabbe disease and other lysosomal storage disorders.

While transplanted neural stem cells (NSCs) have been shown to hold promise for cell replacement in models of a number of neurological disorders, these examples have typically been under conditions where the host cells become dysfunctional due to a cell autonomous etiology, i.e. a 'sick' cell within a relatively supportive environment. It has long been held that cell replacement in a toxic milieu would not likely be possible; donor cells would succumb in much the same way as endogenous cells had. Many metabolic diseases are characterized by this situation, suggesting that they would be poor targets for cell replacement therapies. On the other hand, models of such diseases could prove ideal for testing the capacity for cell replacement under such challenging conditions. In the twitcher (twi ) mouse -- as in patients with Krabbe or globoid cell leukodystrophy (GLD), for which it serves as an authentic model -- loss of galactocerebrosidase (GalC) activity results in the accumulation of psychosine, a toxic glycolipid. Twi mice, like children with GLD, exhibit inexorable neurological deterioration presumably as a result of dysfunctional and ultimately degenerated oligodendrocytes with loss of myelin. It is believed that GLD pathophysiology is related to a psychosine-filled environment that kills not only host oligodendrocytes but theoretically any new cells placed into that milieu. Through the implantation of NSCs into the brains of both neonatal and juvenile/young adult twi mice, we have determined that widespread oligodendrocyte replacement and remyelination is feasible. NSCs appear to be intrinsically resistant to psychosine -- more so in their undifferentiated state than when directed ex vivo to become oligodendrocytes. This resistance can be enhanced by engineering the NSCs to over-express GalC. Some twi mice grafted with such engineered NSCs had thicker white tracts and lived 2-3 times longer than expected. While their brains had detectable levels of GalC, it was probably more significant that their psychosine levels were lower than in twi mice that died at a younger age. This concept of resistance based on differentiation state extended to human NSCs which could similarly survive within the twi brain. Taken together, these results suggest a number of points regarding cellular therapies against degenerative diseases with a prominent cell non-autonomous component: Cell replacement is possible if cells resistant to the toxic environment are employed. Furthermore, an important aspect of successful treatment will likely be not only cell replacement but also cross-correction of host cells to provide them with enzyme activity and hence resistance. While oligodendrocyte replacement alone was not a sufficient treatment for GLD (even when extensive), the replacement of both cells and molecules -- e.g. with NSCs that could both become oligodendrocytes and 'pumps' for GalC -- emerges as a promising basis for a multidisciplinary strategy. Most neurological disease are complex in this way and will likely require multifaceted approaches, perhaps with NSCs serving as the 'glue'.

Psychosine (galactosylsphingosine) is a toxic metabolite that accumulates in globoid cell leukodystrophy (GLD) due to the deficiency of galactocerebrosidase (GALC) activity. This results in subsequent programmed cell death of oligodendrocytes and demyelination in human patients and animal models. We investigated the potential role of insulin-like growth factor-1 (IGF-1) in modifying the apoptotic effect of psychosine in cultured mouse oligodendrocyte progenitor cells (OLP-II). We show that psychosine inhibits the phosphorylation of Akt and Erk1/Erk2 (Erk1/2), which are the main anti-apoptotic pathways of the IGF-1 receptor (IGF-1R). Although IGF-1 sustained phosphorylation of both of these pathways, it provided maximum protection to OLP-II cells from psychosine-induced cell death in a PI3K/Akt-dependent manner. The effects of IGF-1 were dose-dependent and resulted in increased IGF-1R autophosphorylation levels. Although relatively high concentrations of IGF-1 also resulted in the activation of the insulin receptor (IR), its effect was more significant on the IGF-1R.

Globoid cell leukodystrophy (GLD) is a disorder of the central and peripheral nervous systems caused by the deficiency of the lysosomal enzyme galactocerebrosidase (GALC). The pathological changes associated with the disease include accumulation of globoid cells and loss of myelin due to production of psychosine, a toxic metabolite responsible for the apoptosis of oligodendrocytes. While most patients present with symptoms before 6 months of age, older patients are also diagnosed. Treatment at this time is limited to hematopoietic stem cell transplantation in asymptomatic and late-onset patients. GLD occurs naturally in several animal species including mice, dogs, and monkeys. In addition, a transgenic (trs) mouse model of GLD was generated in our laboratory. Trs mice develop symptoms slower than twitcher mice and survive an average of 10 days longer. In this study, we evaluated the therapeutic effects of bone marrow transplantation (BMT) using trs mice. BMT prolonged the life of some treated animals to over one year. After BMT, GALC activity reached 15-20% of normal in brain and near normal values in liver and sciatic nerve. In long-lived transplanted animals psychosine levels were normalized in the brain and greatly reduced in the sciatic nerve. Staining of brain sections showed more abundant and better quality myelin and near absence of globoid cells. Electron micrographs of sciatic nerves showed reduced endoneurial edema, increased axon density, and abundant onion bulb structures associated with remyelinating axons. Therefore, BMT can ameliorate many of the biochemical and pathological features of GLD. However, additional therapies may be required to completely correct the features of this disease.

Globoid-cell leukodystrophy (GLD) is an autosomal recessive lysosomal storage disorder caused by mutations in the galactosylceramidase (GALC) gene. Infantile GLD has a lethal course with severe cerebral demyelination that progresses to death by 2 years of age. In the current study twitcher mice, an authentic murine model of infantile GLD, were given intracranial injections of either recombinant adeno-associated virus serotype 2 encoding the murine Galc cDNA (AAV2-GALC) or the same genome pseudotyped with AAV5 capsid proteins (AAV2/5-GALC) on day 3 of age. The group injected intracranially with AAV2/5-GALC had approximately 25-fold greater than normal Galc levels in the brain, while AAV2-GALC-injected animals had 28% normal levels. The average life expectancy of twitcher mice ( approximately 38 days) was significantly (P < 0.0001) increased to 48 and 52 days for the AAV2-GALC- and AAV2/5-GALC-treated groups, respectively. The AAV2/5-GALC group performed significantly better in a battery of behavioral tests compared to untreated, AAV2-GFP-treated, or AAV2-treated twitcher animals. This longitudinal study demonstrated that AAV2/5-GALC-mediated gene therapy resulted in higher levels of Galc expression and slowed the neurologic deterioration more completely than AAV2-GALC in the murine model of globoid-cell leukodystrophy. However, the clinical improvements, as assessed by behavioral tests and life span, were only modest.

Background: Infantile Krabbe's disease produces progressive neurologic deterioration and death in early childhood. We hypothesized that transplantation of umbilical-cord blood from unrelated donors before the development of symptoms would favorably alter the natural history of the disease among newborns in whom the disease was diagnosed because of a family history. We compared the outcomes among these newborns with the outcomes among infants who underwent transplantation after the development of symptoms and with the outcomes in an untreated cohort of affected children., Methods: Eleven asymptomatic newborns (age range, 12 to 44 days) and 14 symptomatic infants (age range, 142 to 352 days) with infantile Krabbe's disease underwent transplantation of umbilical-cord blood from unrelated donors after myeloablative chemotherapy. Engraftment, survival, and neurodevelopmental function were evaluated longitudinally for four months to six years., Results: The rates of donor-cell engraftment and survival were 100 percent and 100 percent, respectively, among the asymptomatic newborns (median follow-up, 3.0 years) and 100 percent and 43 percent, respectively, among the symptomatic infants (median follow-up, 3.4 years). Surviving patients showed durable engraftment of donor-derived hematopoietic cells with restoration of normal blood galactocerebrosidase levels. Infants who underwent transplantation before the development of symptoms showed progressive central myelination and continued gains in developmental skills, and most had age-appropriate cognitive function and receptive language skills, but a few had mild-to-moderate delays in expressive language and mild-to-severe delays in gross motor function. Children who underwent transplantation after the onset of symptoms had minimal neurologic improvement., Conclusions: Transplantation of umbilical-cord blood from unrelated donors in newborns with infantile Krabbe's disease favorably altered the natural history of the disease. Transplantation in babies after symptoms had developed did not result in substantive neurologic improvement., (Copyright 2005 Massachusetts Medical Society.)

Globoid cell leukodystrophy (GLD) or Krabbe disease is a neurodegenerative disorder caused by a deficiency of galactocerebrosidase (GALC) activity. GALC is required for the lysosomal degradation of galactosylceramide, psychosine, and possibly other galactolipids. This process is extremely important during active myelination. In the absence of functional GALC, psychosine accumulates, resulting in the apoptotic death of myelin-producing cells. While most patients are infants who do not survive beyond 2 years of age, some older patients are also diagnosed. Hematopoietic stem cell transplantation has proven to have a positive effect on the course of some patients with late-onset Krabbe disease. Murine models of this disease provide an excellent opportunity to evaluate therapeutic alternatives including gene therapy. In this study we used serotype 1 AAV to express mouse GALC under the control of the human cytomegalovirus promoter. Direct administration of these viral particles into the brains of neonatal mice with GLD resulted in sustained expression of GALC activity, improved myelination, attenuated symptoms, and prolonged life span. While this treatment also resulted in significant pathological improvements, the treated mice died with symptoms similar to those of the untreated mice. Additional initiatives may be required to prevent the onset of disease and reverse the course of the disease in animal models and human patients.

We describe three brothers suffering from Krabbe's disease with onset in the fifth decade. The proband showed a complete deficiency of leukocyte enzyme galactocerebrosidase and was found to be heterozygous for two previously described mutations: G > A809 and 502T/del consisting of a 30 kb deletion. In all three brothers the neurological examination showed features of asymmetrical peripheral neuropathy associated with pyramidal signs and the electrophysiological examination showed a generalized slowing of nerve conduction velocities. Two patients died at 59 and 61 years of age due to respiratory failure. Both the proband and his brother underwent a sural nerve biopsy. In the former the most striking finding was the presence of uniformly thin myelin sheaths without evidence of demyelination; a complete absence of fibers was found in the latter. Our findings confirm that peripheral neuropathy may be the presenting feature of late-onset Krabbe's disease. Hypomyelination rather than demyelination may represent the distinguishing pathological finding of this condition.

Galactocerebrosidase (GALC) is deficient in all tissues from human patients and animal models with globoid cell leukodystrophy (GLD) or Krabbe disease. The deficiency results in decreased lysosomal catabolism of certain galactolipids including galactosylceramide and psychosine that are synthesized maximally during myelination. According to current theories, the accumulation of psychosine in humans and animals with GLD induces oligodendrocyte degeneration and myelination ceases. Transduction of oligodendrocytes from twitcher mice with a retroviral vector containing the GALC cDNA can correct the enzyme deficiency in these cells. Our data show that twitcher astrocytes and oligodendrocytes can internalize exogenous GALC, as well as donate the enzyme to the mutant glial cells. Antibodies against human GALC localized the GALC antigen in retrovirally transduced cells and cells receiving enzyme via cell to cell secretion and uptake to the lysosomal fraction. In fact immunocytochemical studies in transduced oligodendrocytes revealed that the GALC colocalizes in vesicles lysosomal-associated membrane protein-2 (LAMP2) (+). Moreover, labeling cells with anti-GALC and a marker for oligodendrocytes demonstrated that, upon differentiation, transduced, twitcher oligodendrocytes attained the normal branched process configuration, while untransduced cells show only abnormal morphology. Phenotype correction in mutant oligodendrocytes has also been observed after enzyme transfer. These studies indicate that GALC activity supplied to cultured oligodendrocytes from twitcher mice by different methods can correct the pathological phenotype of these cells., (Copyright 2001 Academic Press.)

Globoid cell leukodystrophy (Krabbe disease) is a severe leukodystrophy caused by mutations in the galactocerebrosidase (GALC) gene leading to extremely low (less than 5% of normal activity) GALC activity. Human patients include primarily severely affected infants as well as patients with a later onset of symptoms. The infants usually die before 2 years of age, but it is difficult to predict the clinical course in older patients. In addition to these patients, additional individuals identified in this laboratory have 10--20% of normal GALC activity measured in accessible tissues. These individuals have a wide range of clinical presentations involving neurological degeneration. On molecular analysis of the GALC gene they all have three or more mutations considered to be normal polymorphisms resulting in amino acid changes in the two copies of the GALC gene. In order to investigate the role these amino acid changes may play on clinical, biochemical, and pathological findings, a new transgenic mouse was generated by homologous recombination. After preliminary studies determined what effect each amino acid change had on mouse GALC activity in transient transfection experiments, mice containing a cysteine residue at codon 168 instead of histidine (H168C) were produced. These mice developed symptoms, but they were delayed by 10--15 days from the well-characterized twitcher (twi) mouse. They accumulated psychosine slightly slower than twi mice, showed pathological changes less severe than twi mice in the central and peripheral nervous systems, and live about 15 days longer than twi mice. They have large litters and will play a role in therapy trials using new procedures currently under development., (Copyright 2001 Academic Press.)

Globoid cell leukodystrophy (GLD) or Krabbe disease is an autosomal recessively inherited neurological disease caused by mutations in the gene coding for the lysosomal enzyme galacto-cerebrosidase (GALC). GALC is responsible for the degradation of specific galactolipids, including several that are important in the production of compact, stable myelin. A failure to adequately degrade galactosylceramide and psychosine (galactosylsphingosine) results in the characteristic pathological findings observed in tissue from humans and animals affected with GLD. These galactosphingolipids are normally synthesized during active myelination, and psychosine accumulates in individuals with very low GALC activity. Psychosine is highly toxic to the myelin-forming oligodendrocytes, causing their death and the paucity of myelin found on autopsy. While most human patients present with symptoms before six months of age and die before 18 months of age, older children and adults can also be diagnosed with GLD[1,2]. The cloning of both the human GALC cDNA and the GALC gene opened the way for the identification of mutations causing GLD in humans and animals and the development of novel strategies to treat this severe and fatal disease[3]. The pheno-typic differences between human patients result from the wide range of mutations identified, as well as additional unknown factors. Treatment of late-onset patients and pre-symptomatic individuals (identified either because prenatal testing was not requested or a fetus predicted to be affected was not aborted) by hemato-poietic stem cell transplantation (HSCT) resulted in a less severe phenotype than was predicted and, in some cases, a significant delay in the onset of symptoms[4]. Attempts to treat this disorder by in utero HSCT have not been successful[5].GLD in dogs

We present the MR imaging findings in four patients (two pairs of siblings from two unrelated families) with adult Krabbe disease. In the first family, clinical presentation mimicked familial spastic paraplegia. Their MR images showed selective, increased signal intensity on T2-weighted sequences along the corticospinal tracts, most prominently in the proband and barely detectable in her brother. Proton MR spectroscopy showed increased choline and myo-inositol in the affected white matter. In the second family, the clinical presentation differed in that the signs of pyramidal tract involvement were asymmetrical, with concomitant asymmetry on MR images in one. In adults, Krabbe disease may present on MR imaging with selective pyramidal fiber involvement.

Krabbe disease or globoid cell leukodystrophy is a disorder involving the white matter of the peripheral and central nervous systems. Mutations in the gene for the lysosomal enzyme galactocerebrosidase (GALC) result in low enzymatic activity and decreased ability to degrade galactolipids found almost exclusively in myelin. The pathological changes observed, including the presence of globoid cells and decreased myelin, appear to result from the toxic nature of psychosine and accumulation of galactosylceramide that cannot be degraded due to the GALC deficiency. Over 60 mutations have been identified in this gene. The great majority are disease-causing; however, a few are considered polymorphisms. While most patients present with symptoms within the first 6 months of life, others present later in life including adulthood. Even patients with the same genotype can have very different clinical presentations and course. The reason for this is not known. Treatment at this time is limited to hematopoietic stem cell transplantation that appears to slow the progression of the disease and improve the magnetic resonance images. Studies using stem cells and viral vectors to transduce transplantable cells are under way in model systems. In culture, oligodendrocytes from the twitcher mouse model can assume a normal appearance after differentiation if GALC activity is provided via viral transduction or uptake from donor cells. Therefore continued myelination and/or remyelination in patients will require supplying GALC activity by transplanted cells or viral vectors to still functional endogenous oligodendrocytes or transplantation of normal oligodendrocytes or stem cells that can differentiate into oligodendrocytes. Using the animal models these options can be explored., (Copyright 2000 Academic Press.)

Purpose: This study was designed to examine the use of quantitative magnetization transfer imaging (MTI) in naturally occurring globoid cell leukodystrophy (GLD) in the Cairn terrier., Method: A model of GLD was established via a breeding colony, and a total of seven animals were studied with MTI, including two dogs with GLD, one of which underwent whole-body irradiation (725 cGy) and bone marrow transplantation from a genotypically normal littermate. The remaining dogs served as untreated, irradiated, and unirradiated controls., Results: Region-of-interest (ROI) analysis of the MTI showed a decrease in MT ratio (MTR) in the internal capsule of the untreated/affected dog compared with age-matched controls but revealed similar results in the two other study animals. On MT contour plotting, inside-to-out gradients of MTR mimicked the demyelination pathology of the disease in the untreated/affected dog., Conclusion: MT contour plotting demonstrated patterns of MT abnormality in the untreated/affected dog that were consistent with histopathology, establishing a clear relationship between pathology-proven demyelination and MTR as well as a striking contrast to the patterns of radiation damage.

Background: Krabbe disease, or globoid cell leukodystrophy, is an autosomal recessive disorder caused by the deficiency of galactocerebrosidase (GALC) activity. Although most cases are diagnosed in infancy and show a fatal outcome in childhood, adult patients have been identified, showing progressive spastic hemiparesis to tetraparesis, followed by optic atrophy, dementia, and neuropathy. The disease can be diagnosed by detecting the deficiency of GALC activity (less than 5% of normal) in any available tissue sample. The cloning of the human GALC gene allowed the molecular characterization of newly diagnosed patients. More than 75 disease-causing mutations and polymorphisms in this gene have been identified., Objective: To describe a 28-year-old woman with Krabbe disease, correlating clinical and biochemical abnormalities to a novel mutation on the GALC gene., Methods: Clinical investigation was enriched by neurophysiological and neuroimaging data. The activity of GALC was assayed in white blood cells using radiolabeled natural substrate. Genomic DNA was isolated from peripheral blood, and the GALC gene was sequenced. The mutated gene was expressed and GALC activity was measured in transfected COS-1 cells., Results: The patient had progressive and bilateral amaurosis starting at 8 years of age. Although she was experiencing weakness in all her extremities, her intellect remained intact. She was found to be homozygous for a previously unreported missense mutation (T1886G), which leads to low, but not totally deficient, GALC activity., Conclusions: Expression of this mutation in COS-1 cells using the pcDNA3 expression vector (Invitrogen, Carlsbad, Calif) resulted in low, although not null, GALC activity, which can explain the protracted clinical course in this patient. Patients carrying the mutation described herein might be potential candidates for therapeutic trials, such as bone marrow transplantation or gene therapy.

Krabbe disease or globoid cell leukodystrophy (GLD) is an autosomal recessive disorder resulting from the defective lysosomal hydrolysis of specific galactolipids found primarily in myelin. This leads to severe neurological symptoms including seizures, hypotonia, blindness, and death, usually before 2 years of age in human patients. In addition to human patients, several animals, including dog, mouse, and monkey, have the same disease caused by a deficiency of galactocerebrosidase (GALC) activity. In this article we describe studies in cairn and West Highland white terriers (WHWT) affected with GLD. Through a screening test based on the molecular defect found in these breeds, over 50 cairn terrier carriers have been identified and a colony of five carrier dogs has been established. Affected dogs from this colony plus an affected WHWT were available for study. An affected WHWT was evaluated by magnetic resonance imaging at 6 and 11 months of age and pronounced changes in the T-2 weighted fast spin-echo images were found. Biochemical and pathological evaluation of the same dog after euthanasia at 12 months of age showed a large accumulation of psychosine in the brain and white matter filled with globoid cells. Some comparisons were made to younger affected and carrier dogs. Studies have shown successful transduction of cultured skin fibroblasts from an affected dog and normal canine bone marrow using a retroviral vector containing the human GALC cDNA. Successful treatment of this canine model will lead to studies in some humans with GLD.

Globoid cell leukodystrophy, or Krabbe disease, is a severe disorder of the peripheral and central nervous system myelin caused by deficient galactocerebrosidase (GALC) activity. This autosomal recessive disease affects humans and animals including dogs, mice, and rhesus monkeys. Cloning of the human and animal GALC genes opened opportunities for therapeutic trials using animal models. We describe the clinical, pathologic, and biochemical features of the affected rhesus monkey. Affected monkeys had very low GALC activity and a two base pair deletion in both copies of the GALC gene. Clinical signs of tremors, hypertonia, and incoordination led to humane euthanasia by 5 months of age. At necropsy, peripheral nerves were enlarged. Microscopically, the cerebral, cerebellar, and spinal cord white matter was infiltrated with periodic acid-Schiff-positive multinucleated globoid cells, and there was a striking lack of myelin. Peripheral nerve fibers were decreased in number and separated by Alcian blue- and safranin O-positive material. Myelin sheaths were greatly diminished. Lipid analysis of brains of 12-day-old and 158-day-old affected monkeys revealed a great excess of psychosine in white matter. The rhesus monkey model will be especially useful for exploring treatment options, including prenatal bone marrow transplantation and various approaches to gene therapy.

A six-month-old West Highland white terrier with progressive, multifocal neurological disease was diagnosed with canine globoid cell leucodystrophy (GCL). Magnetic resonance imaging (MRI) of the brain was performed, as well as electrophysiological testing (including brainstem auditory evoked response, peripheral nerve conduction velocity, repetitive stimulation, F wave analysis and electromyography). MRI findings were consistent with diffuse, symmetrical white matter disease. Electrodiagnostic testing revealed evidence of peripheral neuropathy and an abnormal brainstem auditory evoked response. These observations were consistent with the pathological changes in central and peripheral white matter described for canine GCL, and resembled what has been described in human patients. It is believed that the tests may raise the suspicion of GCL in dogs and may aid in monitoring disease progression.

Background: Globoid-cell leukodystrophy is caused by a deficiency of galactocerebrosidase, which results in progressive central nervous system deterioration. We investigated whether allogeneic hematopoietic stem-cell transplantation can provide a source of leukocyte galactocerebrosidase and thereby prevent the decline of central nervous system function in patients with the disease., Methods: Five children with globoid-cell leukodystrophy (one with the infantile type and four with late-onset disease) were treated with allogeneic hematopoietic stem-cell transplantation. Measurement of leukocyte galactocerebrosidase levels, neurologic examinations, neuropsychological tests, magnetic resonance imaging of the central nervous system, cerebrospinal fluid protein assays, and neurophysiologic measurements were performed before and after transplantation, with follow-up ranging from one to nine years., Results: Engraftment of donor-derived hematopoietic cells occurred in all patients and was followed by restoration of normal leukocyte galactocerebrosidase levels. In the four patients with late-onset disease, the central nervous system deterioration was reversed, and in the patient with the infantile form of the disease, signs and symptoms have not appeared. Magnetic resonance imaging showed a decrease in signal intensity in the three patients with late-onset disease who were assessed both before and after transplantation. Abnormalities in cerebrospinal fluid total protein levels were corrected in three patients with late-onset disease and substantially reduced in the patient with the infantile form., Conclusions: Central nervous system manifestations of globoid-cell leukodystrophy can be reversed by allogeneic hematopoietic stem-cell transplantation.

Sixty-four unrelated patients with infantile Krabbe disease (globoid cell leukodystrophy, GLD) of Dutch (n = 41) or other European origin (n = 23) were screened for the presence of a large 30 kb deletion starting in intron 10 (IVS10del30 kb), a base substitution 1538T(T513M) and a polymorphism, 502T. The deletion and the T513M mutation were present in 52% and 8.5%, respectively, of the 82 GALC alleles of the Dutch patients. The 502T polymorphism, which had an allele frequency of 5.3% in a Dutch control panel, occurred in 65% of the GLD alleles. Analysis of patients and both parents in 26 of the families showed that del30 kb was invariably associated with 502T. However, 502T was also present on 40% of the GLD alleles with an as yet unidentified mutation, which is 7.5 times higher than its frequency in controls. This suggests that besides del30 kb at least one other relatively frequent mutation has arisen on the 502T GALC allele. A relatively high incidence of del30 kb was also found in 23 other European (non-Dutch) patients (allele frequency 35%), but T513M did not occur in this group. Practical examples described in this report illustrate the potential usefulness of mutation analysis in many families with Krabbe disease for heterozygote detection and prenatal diagnosis.

Krabbe disease or globoid cell leukodystrophy (GLD) is a severe lysosomal disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. This deficiency results in the insufficient catabolism of several galactolipids that are important in the production of normal myelin. Since the cloning of the human GALC cDNA and gene many disease-causing and polymorphic changes have been identified. This autosomal recessive disease has been reported to occur in several animal species, and recently the murine and canine GALC genes have been cloned. We now describe the cloning of the GALC cDNA and gene from the rhesus monkey and the identification of the mutation causing GLD in this species. The nucleotide sequence of the coding region and the gene organization were nearly identical to human. The deduced amino acid sequence of the monkey GALC was compared to the human, dog, and mouse, and it was found to be 97, 87, and 83% identical, respectively. The mutation causing GLD in the rhesus monkey is a deletion of AC corresponding to cDNA positions 387 and 388 in exon 4. This results in a frame shift and a stop codon after 46 nucleotides. A rapid method to detect this mutation was developed, and when 45 monkeys from this colony were tested, 22 were found to be carriers. The availability of this nonhuman primate model of GLD will provide unique opportunities to evaluate treatment for this severe disease.

To date, in utero bone marrow transplantation (BMT) has had limited success, largely because of poor donor engraftment. The poor engraftment is probably the result of performing the procedure late in gestation after significant fetal immunocompetence has developed and/or transplanting insufficient numbers of donor hematopoietic stem cells for competing successfully with ongoing fetal hematopoiesis. To overcome these problems, we performed in utero BMT on a fetus with globoid cell leukodystrophy during the first trimester of gestation using selected paternal bone marrow stem (CD34+) cells. CD34 selection allowed a substantially greater number of stem cells to be transplanted. Although the fetus died 7 weeks after the procedure (during the 20th week of gestation), full donor engraftment was established. Moreover, the cause of death appeared to be overwhelming donor engraftment and leukostasis with paternal myeloid cells infiltrating most tissues. The ability of in utero BMT to produce this degree of engraftment provides great promise for the use of this approach in the treatment of a variety of inherited disorders that can be diagnosed prenatally.

Galactocerebrosidase (GALC) is a lysosomal beta-galactosidase responsible for the hydrolysis of the galactosyl moiety from several galactolipids, including galactosylceramide and psychosine. The deficiency of this enzyme results in the autosomal recessive disorder called Krabbe disease. It is also called globoid cell leukodystrophy (GLD), because of the characteristic storage cells found around cerebral blood vessels in the white matter of affected human patients and animal models. Although most patients present with clinical symptoms before 6 months of age, older patients, including adults, have been diagnosed by their severe deficiency of GALC activity. More than 40 mutations have been identified in patients with all clinical types of GLD. While some mutations clearly result in the infantile type if found homozygous or with another severe mutation, it is difficult to predict the phenotype of novel mutations or when mutations are found in the heterozygous state. A high incidence of polymorphic changes on apparent disease-causing alleles also complicates the interpretation of the effects of mutations. The detection of mutations has greatly improved carrier identification among family members and will permit preimplantation diagnosis for some families. The molecular characterization of the naturally occurring mouse, dog, and monkey models will permit their use in trials to evaluate different modes of therapy.

A 53-year-old man was diagnosed 8 years earlier with globoid cell leukodystrophy (GLD, Krabbe disease) by his severe deficiency of galactocerebrosidase (GALC) activity. He was found to have eight nucleotide changes on the two copies of his GALC gene, including two in the leader sequence, four considered polymorphisms, and two unique mutations.

Globoid cell leukodystrophy, or Krabbe disease, is a severe, autosomal recessive disorder resulting from a deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of certain galactolipids, including galactosylceramide and psychosine. In addition to the human patients, there are several naturally occurring animal models for this disease, including the twitcher mouse, West Highland White terriers (WHWT), and Cairn terriers. All species have deficient GALC activity and have the characteristic pathological findings in the nervous system. We now describe the cloning of the canine GALC cDNA and the identification of the disease-causing mutation in both terrier breeds. The 2007-bp open reading frame is 88% identical to that in human, and the deduced amino acid sequence is about 90% identical. However, the 3'-untranslated region is about 1 kb shorter than that in the human. Two nucleotide changes were found in affected dogs, an A to C transversion at cDNA position 473 (Y158S) and a C to T transition at position 1915 (P639S). Expression studies in COS-1 cells demonstrated that the A to C change at 473 is the disease-causing mutation. A rapid test for the identification of the genotype at that position has been developed, and over 100 WHWT and Cairn terriers have been screened. This will allow breeders to mate their dogs selectively and will permit the establishment of a colony of dogs for use in therapy trials.

Infantile Krabbe disease is a severe, fatal autosomal recessive disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. It is relatively common in two separate inbred communities in Israel. In the Druze community in Northern Israel and two Moslem Arab villages located near Jerusalem the incidence of Krabbe disease is about 1 in 100-150 live births. With our cloning of the GALC gene, mutation analysis of these populations was undertaken. The Moslem Arabs were homozygous for two mutations in the GALC gene; a T-to-C transition at CDNA position 1637 (counting from the A of the initiation codon), which is considered a polymorphism and a G-to-A transition at position 1582, which changes the codon for aspartic acid to one for asparagine. The Druze patients are homozygous for a T-to-G transversion at position 1748, which changes the codon for isoleucine to one for serine. Expression studies confirmed the deleterious nature of these mutations. The development of a simple polymerase chain reaction (PCR) amplification and restriction enzyme digestion method to identify these alleles will lead to accurate carrier testing and improved genetic counseling for interested individuals in these communities.

Globoid cell leukodystrophy (GLD) of Krabbe disease results from mutations in the galactocerebrosidase (GALC) gene. Previously, we had identified a large deletion in the GALC gene together with a C to T polymorphism at cDNA position 502 in a significant number of cases of infantile Krabbe disease; however, the deletion breakpoint had not been found. In this paper we show that the deletion is approximately 30 kb starting near the middle of the 12 kb intron 10, and includes all of the coding region through exon 17 plus an additional 9 kb. The deletion junction contains a 4 bp direct repeat and is preceded by sequence identified as belonging to the Alu family of interspersed repetitive elements. Using genomic DNA and a PCR-based test to detect normal and deleted sequences at that location, a large number of patients with all clinical types of GLD were analyzed. Of 21 infantile patients found to be heterozygous for the 502T polymorphism reported previously, 15 had the deletion, one could not tested and five, including a Hmong child, did not have the deletion. Sixteen other infantile patients previously tested were found to be either homozygous (10) or heterozygous (6) for the deletion. In addition, five patients with juvenile and adult GLD were found to be heterozygous for the deletion. In every case tested, the deletion was always found on the same allele as the 502T polymorphism. However, other disease-causing mutations have been found on the 502T allele. With careful genotype analysis these families can receive improved genetic information including patient and carrier identification and preimplantation diagnosis.

Galactocerebrosidase (GALC) activity is deficient in all patients with globoid cell leukodystrophy (GLD). While most patients have the severe infantile form of this autosomal recessive disorder (Krabbe disease), patients up to 50 years of age have been diagnosed in this laboratory. With the cloning of the GALC cDNA and availability of information regarding the gene organization, patients can be evaluated for their disease-causing mutations. We now report that a large deletion, together with a polymorphic C to T transition at position 502 of cDNA (counting from the A of the initiation codon), is responsible for a large number of disease-causing alleles in patients with Krabbe disease. Of 48 patients evaluated, 10 were found to be homozygous for the 502/del allele, five patients were heterozygous for this allele, 21 patients were heterozygous for the 502 mutation (presence of the deletion could not be confirmed), and one infantile patient was homozygous for the 502 mutation but at least one allele was not deleted. No patient was found to have the deletion without the 502 polymorphism. The delineation of mutations causing infantile Krabbe disease will provide new information regarding structure-function relationships in this multi-subunit enzyme and will improve the identification of patients and carriers in some families.

The gene responsible for Krabbe disease, an autosomal recessive disorder caused by deficiency of galactocerebrosidase (GALC), was localized by multipoint linkage analysis on chromosome 14. Eight mapped dinucleotide repeat polymorphisms were tested for linkage to GALC. Two-point linkage analysis demonstrated close linkage of GALC and D14S48, with Z = 13.69 at theta = 0. Multipoint analysis yielded strong support for this finding, with maximum likelihood for GALC located within 1 cM of D14S48. This analysis also identified markers that clearly flank the GALC locus, as the map order of D14S53-GALC-D14S45 is favored by odds greater than 10(6):1. Additional support for close linkage of GALC and D14S48 comes from the apparent linkage disequilibrium between these two loci in a consanguineous Druze community in Israel. These data localize GALC to 14q24.3-q32.1.

Globoid cell leukodystrophy (Krabbe disease) is an autosomal recessive disorder resulting from the deficiency of galactocerebrosidase (GALC) activity. GALC is responsible for the lysosomal catabolism of galactosylceramide, a major lipid in myelin, kidney and epithelial cells of small intestine and colon. We describe the molecular cloning of human GALC cDNA and its expression in COS-1 cells. Degenerate PCR primers, derived from N-terminal amino acid sequence from the 51 kDa band from human brain, were used to amplify cat testes RNA, and the resulting product was used to screen human testes and brain libraries. Two overlapping clones contained the total protein coding region, while additional clones and PCR amplification were needed to obtain the complete 3' end of the cDNA. The 3795 bp obtained include 47 bp 5' to the initiation start site, 2007 bp of open reading frame (coding for 669 amino acids), and 1741 bp of 3' untranslated sequence. Modification of the sequence surrounding the initiation codon to one more favorable for expression, resulted in a 6-fold increase in GALC activity in transfected COS-1 cells. The isolation of this clone will permit investigations into the causes for GALC deficiency in humans and available animal models, development of more accurate tests for patient and carrier identification, and evaluation of methods for effectively treating GALC deficiency, initially using the animal models.