History and Disease Definitions:
Krabbe disease is an autosomal recessive disorder also known as globoid cell leukodystrophy (Krabbe, 1916). By definition, leukodystrophies are diseases in which the white matter of the brain degenerates, often due to demyelination (Suzuki and Suzuki, 1970). Krabbe disease is characterized pathologically by the presence of multinucleated macrophages in the brain with a distinct morphology (see Figure 3) and a high galactolipid concentration (Suzuki and Suzuki, 1970). Krabbe Disease is also classified as a lyosomal storage disorder because the disease is rooted in the deficiency of the lysosomal hydrolytic enzyme β-galactocerebrosidase. This deficiency pervades all cells in the body, but adverse consequences of the absence of β-galactocerbrosidase are observed largely in cells involved in myelin metabolism (Suzuki and Suzuki, 1970).
Dr. Knud Krabbe first encountered what would later be coined Krabbe Disease in 1913 when treating a patient who died at thirteen months old at a hospital in Copenhagen, Denmark (Krabbe, 1916). He diagnosed the patient with “diffuse brain sclerosis with perivascular necrosis of the medullary substance,” noting the neurodegenerative behaviors observed and the patient’s muscle spasms and posture of decerebrate rigidity. Krabbe was even more surprised, however, by the unusual extent of destruction of the white matter throughout the brain and spinal cord that he observed during the autopsy and subsequent analysis (Krabbe, 1916). Krabbe also identified large and irregularly shaped multinucleated cells within the glial fibers of the brain that he had never previously observed. Krabbe called these “empty-granule cells” because he was surprised by their lack of fatty granules. Krabbe also noted that he could not find any of the typical leukocytes normally seen in the brain (1916).
Dr. Krabbe was compelled to investigate this disease further because he had also cared for this patient’s older sister who had suffered from the same symptoms and disease course; she had died at one year of age. The only difference in disease characteristics identified post-morten was that the young girl’s brain had much higher concentrations of “fatty substances (Krabbe, 1916).” Krabbe discussed these findings with a colleague, Dr. Bloch, who had also cared for two siblings with nearly identical pathology and disease course in 1907 (Krabbe, 1916).
Krabbe grouped these four cases with another isolated case based once again on disease pathology. He noted that all of the patients experienced symptom onset between 4-6 months old, and that the disease seemed to have an inherited component. Most significantly, Krabbe noted the extreme white matter degradation accompanied by the consistent presence of “empty-granule cells” and the consistent absence of normal leukocytes and other inflammatory cells. These observations collectively caused Krabbe to reconsider his initial thoughts of an inflammatory disease. Krabbe also noted that while the inherited neurodegenerative component of the disease resembled Tay-Sachs, the pathology of the disease was very different. Krabbe thus identified this disease as a new form of “early infantile familial diffuse brain sclerosis (Krabbe, 1916).” It was not until 1970, however, that Suzuki and Suzuki identified β-galactocerebrocidase as the cause of the disease (Suzuki and Suzuki, 1970).
Disease-free metabolic context
β-galactocerbrosidase (E.C. 188.8.131.52) is a lysosomal enzyme that normally functions in myelin biosynthesis and myelin breakdown. Myelin (Figure 1) is composed of about 70% lipids and 30% myelin associated proteins. It encapsulates axons throughout the Central Nervous System (CNS) and Peripheral Nervous System (PNS); it is secreted by oligodendrocytes (CNS) and Schwann cells (PNS). Myelin protects neurons from axonal injury and promotes rapid action potential propagation by decreasing the capacitance of the axonal membrane, thereby increasing electrical resistance and insulating the axon to prevent loss of current (Hartline, 2008).
Approximately 27% of myelin lipids are composed of galactocerebroside (also called galactosylceramide) and sulfide, a sulfated derivative of galactocerebroside (Norton and Cammer, 1984). Sulfatide, galactocerebroside, and monogalactosyldiglyceride (a minor myelin component) are synthesized (Figure 2A) from UDP-galactose and myelin precursors ceramide, sphingosine, and diglyceride through a hexosyltransferase reaction catalyzed by UDP-galactosyltrasnferase (E.C. 184.108.40.206). While the resulting galactocerebroside, sulfatide, and monogalactosyldiglyceride are readily incorporated into myelin, the product of the UDP- galactosyltrasnferase catalyzed hexosyltransfer to sphingosine forms psychosine, a toxic metabolite (Igisu and Suzuki 1994). In healthy oligodendrocytes and Schwann cells, psychosine (Figure 2C) is quickly hydrolyzed by β-galactocerbrosidase to re-form sphingosine and galactose. β-galactocerbrosidase is also essential to myelin catabolism (Figure 2B) by breaking down sulfatide, galactocerebroside, and monogalactosyldiglyceride into galactose and the appropriate lipid precursor (Norton and Cammer, 1984). While sulfatide, galactocerebroside, and monogalactosyldiglyceride are not toxic compounds, the sheer accumulation of these metabolites is believed to contribute to psychosine-induced oligodendrocyte, Schwann cell, and microglia toxicity (Norton and Cammer, 1984).
About 1 in 100,000 individuals are diagnosed with Krabbe Disease (Duffner et al., 2012). The Durze population in Northern Israel and the Muslim Arab population surrounding Jerusalem historically have a higher prevalence of Krabbe Disease at 1 in 130 individuals (Zlotogora et al. 1991). There are four classifications of Krabbe disease based on the age at which symptoms first develop.
1. Early infantile form arises between three and six months of age—the predominant symptoms are blindness, missed developmental milestones, failure to thrive (stunted growth and poor appetite), a rigid posture due to neurological damage in which the baby remains on his or her back with the limbs rotated inwardly (called decerebrate rigidity), and frequent muscle spasms leading to muscular rigidity (Lyon et al. 1991). Early infantile Krabbe Disease is the most common disease phenotype (more than 90% of patients experience early infantile onset) and most severe form of the condition—death usually occurs between ages one and three (Duffner et al. 2012).
2. Late infantile form is classified by onset of symptoms between seven months and one year of age, and 3. juvenile form is classified by onset of symptoms in one through ten year old children. Symptoms of both of these forms of Krabbe disease include decreased cognitive function, overall muscle weakness, loss of muscle control, optic nerve and muscle disintegration, and vision loss (Lyon et al. 1991).
4. Adolescent/adult onset (individuals experience symptoms after 11 years old) is extremely rare and progresses the most slowly—it is characterized by muscle stiffness and weakness, loss of bladder control, and occasionally paralysis. Late symptom onset is correlated with slower disease progression and increased survival rates (Duffner et al. 2012). This classification of Krabbe Disease is also the newest– the first case of adult onset was documented in the early 1980s (Thomas et al., 1984).
Means of Identification:
In the absence of a known family history of Krabbe Disease, testing for Krabbe Disease is generally done in response to any combination of the aforementioned symptoms. The New York, New Jersey, Missouri, Illinois, and New Mexico Departments of Health all conduct mandatory newborn screening for Krabbe Disease as part of a battery of newborn tests (NPR 2013). Screening for Krabbe Disease begins with obtaining a blood sample from the infant. The patient’s leukocytes are isolated and tested for β-galactocerebrosidase activity by incubating an aliquot of the sample with a fluorgenic substrate called HMGal on a 96-well plate; when β-galactocerebrosidase hydrolyzes the HMGal, the fluorophore 6-decanoylamino-4- methylumbelliferone is released and detected (Patent). Subsequent analysis by MS/MS is also completed. While an absolute enzymatic activity value is calculated (typically around 1.20 nmol/h/mg protein), results are typically assessed and reported in terms of percent daily mean activity of (DMA) of all samples processed that day to compensate for daily variability in processing. If the calculated %DMA is less than or equal to 20%, the sample is re-tested immediately. If the re-test yields a %DMA ranging from 8-12%, the sample is sequenced to test for the most common GALC mutation: a 30 kb deletion from the long arm of chromosome (Kemper et al., 2010) . If the re-test yields a %DMA less than 8%, the newborn’s pediatrician is notified that patient has a positive screening for Krabbe Disease and the sample is sequenced to test for the 30 kb deletion. Subsequent screening for alternative GALC mutations may be completed if the 30 kb deletion is not present. This screening procedure is also applied to all patients who visit the pediatrician due to symptoms that may be indicative of Krabbe disease, regardless of age of onset (Kemper et al., 2010).
Additional diagnostic procedures for Krabbe Disease include assessment of cerebrospinal fluid protein levels through a spinal tap and of pathophysiology of the nervous system tissue biopsy. Symptomatic patients with early infantile Krabbe Disease typically have elevated cerebrospinal fluid (CSF) protein levels (183 mg/dL to 422 mg/dL); elevated CSF protein levels are less common in patients with late infantile, juvenile, and adult/adolescent onset (Duffner et al., 2009). Globoid cells are multinucleated macrophages derived from microglia that are 15-20 nm in diameter; unlike healthy macrophages, they are unreactive to glial fibrillary acidic protein (Krabbe, 1916).
Physicians may also utilize MRIs to diagnose Krabbe Disease and gauge the severity of the case. Early-onset Krabbe Disease MRIs typically yield brain atrophy, deep grey nuclei, cerebellar white matter, and abnormal pyramidal tracts (Figure 4A); late infantile onset and juvenile onset are characterized by parieto-occipital white matter and posterior corpus callosum; cerebellar white matter and deep grey nuclei are not observed (Figure 4B; Loes et al., 1999).
Finally, nerve conduction studies in one motor and one sensory nerve in both an upper and a lower extremity are sensitive indicators of Krabbe Disease for individuals with early infantile onset. Distal latency, conduction velocity, presence and amplitude of response, and F-wave latency are assessed in each nerve tested. Peripheral neuropathy studies are provide less clinical insight in patients with late infantile, juvenile, and adult/adolescent onset (Duffner et al., 2009).
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