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|Coordinate||98,234,266 bp (GRCm38)|
|Base Change||T ⇒ C (forward strand)|
|Synonym(s)||Gacy, A930008M05Rik, 2310068B06Rik, galactocerebrosidase|
|Chromosomal Location||98,202,304-98,259,459 bp (-)|
|MGI Phenotype||Homozygotes for spontaneous and targeted mutations exhibit tremors, progressive weakness, wasting, both central and peripheral demyelination, massive accumulation of galactosylceramide, abnormal macrophages, and death by 4 months of age.|
|Amino Acid Change||Asparagine changed to Serine|
|Institutional Source||Beutler Lab|
|Gene Model||predicted gene model for protein(s): [ENSMUSP00000021390]|
AA Change: N295S
|Predicted Effect||probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00)
|Phenotypic Category||Autosomal Semidominant|
|Alleles Listed at MGI|
|Mode of Inheritance||Autosomal Semidominant|
|Last Updated||2017-05-25 3:29 PM by Katherine Timer|
|Record Created||2015-12-01 10:28 AM by Emre Turer|
The Crabby2 phenotype was identified among N-ethyl-N-nitrosourea (ENU)-mutagenized G3 mice of the pedigree R1763, some of which showed weight loss compared to wild-type littermates (Figure 1).
|Nature of Mutation|
Whole exome HiSeq sequencing of the G1 grandsire identified 91 mutations. The body weight phenotype was linked to a mutation in Galc: an A to G transition at base pair 98,234,266 (v38) on chromosome 12, or base pair 25,198 in the GenBank genomic region NC_000078 encoding Galc. Linkage was found with an additive model of inheritance, wherein one variant homozygote and five heterozygotes departed phenotypically from seven homozygous reference mice with a P value of 3.313 x 10-7 (Figure 2).
The mutation corresponds to residue 1,014 in the NM_008079 mRNA sequence in exon 8 of 17 total exons.
The mutated nucleotide is indicated in red. The mutation results in an asparagine (N) to serine (S) substitution at position 295 (N295S) in the GALC protein, and is strongly predicted by PolyPhen-2 to be damaging (score = 1.000).
Galc encodes GALC (galactosylceramidase; alternatively, galactocerebrosidase), a member of carbohydrate-active enzymes glycoside hydrolase family 59 (GH59) (1). GALC is a 684-amino acid peptide that has a signal peptide (amino acids 1-24) followed by a triosephosphate isomerase (TIM) barrel (amino acids 41-337), a β-sandwich domain, and a lectin domain (amino acids 572-668) (Figure 3) (1). GALC is an approximately 80 kDa precursor protein. In lysosomes, GALC is processed into two fragments: a 50 kDa N-terminal fragment and a 30 kDa C-terminal fragment (2). The processed protein fragments stabilize the 80 kDa protein, which is the enzymatically active form of the protein (2;3).
The structure of residues 25 to 668 of mouse GALC has been solved (Figure 4; PDB:3ZR6; (1)). Amino acids 25 to 40 comprise two β-strands of the β-sandwich domain before forming the TIM barrel; amino acids 338 to 452 are the remainder of the β-sandwich domain. The TIM barrel has eight parallel β-strands surrounded by α-helices. Similar to other glycosyl hydrolases, the GALC β-sandwich domain comprises two twisted β-sheets. Unique to GALC is the presence of a very long loop that wraps over the top of the TIM barrel. Cys378 within the loop forms a disulfide bond with Cys271 in the TIM barrel. In mouse GALC, a disulfide bond is predicted to form between Cys287 and Cys394. The lectin domain has a similar fold and calcium-binding site as to other glycosyl hydrolayses, including β-glucanase and galectin. However, the GALC lectin domain does not have the catalytic residues found in the β-glucanase family. Residues from the TIM barrel, β-sandwich domain, and lectin domain contribute to the substrate-binding pocket. Human GALC has four putative N-glycosylation sites: Asn284, Asn363, Asn387, and Asn542. In contrast, mouse GALC has six putative N-glycosylation sites: Asn300, Asn379, Asn403, Asn558, Asn601, and Asn645.
The Crabby2 mutation results in an asparagine (N) to serine (S) substitution at position 295 (N295S); residue 295 is within the TIM barrel.
Galc is expressed in mouse brain, liver, and kidney (4). Various isoforms of GALC are differentially expressed in the nervous system (5). An 80-kDa doublet was observed in the central and peripheral nervous systems; 50- and 30-kDa proteins were observed in the cortex and spinal cord, but not sciatic nerve. A 170-kDa protein product was found in the spinal cord, but not the cortex or sciatic nerve; the 170-kDa protein is a putative dimer or oligomer of GALC. Overall, GALC was expressed at lower levels in the sciatic nerve and at higher levels in the cortex and spinal cord. Different glycosylation or phosphorylation patterns may contribute to the GALC expression pattern (5). GALC localizes to the lysosome (2).
GALC is a lysosomal enzyme that hydrolyzes galactosylceramide, psychosine, monogalactosyldiglyceride, and possibly lactosylceramide (Figure 5). Galactosylceramide, psychosine, and monogalactosyldiglyceride are essential for myelination in the nervous system; galactosylceramide is a major component of myelin. Most axons are insulated with myelin sheaths, which allows for the rapid propagation of nerve impulses. Myelin is a multilamellar membrane formed by oligodendrocytes in the central nervous system and by Schwann cells in the peripheral nervous system. During myelination, a glial process wraps around the axon, forming multiple layers of myelin and elongating along the axon. The myelinating glial cells organize the axons into segments: the nodes of Ranvier, paranode, and juxtaparanode. During myelination, oligodendrocyte precursor cells are differentiated into post-mitotic oligodendrocytes. During oligodendrocyte differentiation, the immature oligodendrocytes synthesize large amounts of myelin lipids including galactosylceramide and plasmalogens.
The twitcher mouse model has a spontaneous mutation in Galc that results in nonsense-mediated decay of the Galc transcript (6;7). The twitcher mice are phenotypically normal until approximately postnatal day 20 after which they exhibit progressive neurological phenothypes, including tremors, hindlimb weakness. The twitcher mice also exhibit slowed weight gain and wasting; most mice die by postnatal day 40 (8;9). The twitcher mice exhibit nervous system gliosis, myelin loss in both the peripheral and central nervous systems, and macrophage accumulation, similar to that observed in human Krabbe disease patients (see below) (8;9). The UDP-galactose:ceramide galactosyltransferase activity was normal in the spinal cord of the twitcher mice at postnatal day 15 (10). After postnatal day 15, there was a progressive loss in the galactosyltransferase activity (10). Galactosyltransferase activity remained normal in the kidney at all times examined. The twitcher mice have reduced frequencies of early hematopoietic progenitors and defects in the hematopoietic niche (11).
A second spontatneous Galc mutation (Galctwi-5J) resulting in a glutamic acid to lysine conversion at amino acid 130 causes weakness, stunted weight gain, and generalized tremors after two weeks of age; the Galctwi-5J mice die by approximately four weeks of age (5). The Galctwi-5J mice have gliosis, globoid cells, and psychosine accumulation in the nervous system, but the central nervous system does not exhibit significant demyelination. The peripheral nervous system is hypomyelinated and does not have large diameter axons, indicating primary dysmyelination instead of demyelination.
Galc-deficient (Galc-/-) mice feed normally and exhibit similar rates of growth to wild-type mice 30 days after birth (4). At approximately postnatal day 25 to 30, the Galc-/- mice exhibit tremors and hind leg weakness. The Galc-/- mice are slightly larger than the twitcher mice. The Galc-/- mice gain weight until postnatal day 35 to 40, and then they show progressive weight loss. On average, the Galc-/- mice exhibit lethality at approximately postnatal day 50 to 55. Near the time of death, the Galc-/- mice typically exhibit hind limb paralysis and severe hind limb wasting.
In humans, mutations in GALC lead to global-cell leukodystrophy (GLD; alternatively, Krabbe disease; OMIM: #245200) (12). In most cases, Krabbe disease manifests within the first six months of life, but other Krabbe patients present symptoms later, even into adulthood (13). Krabbe patients exhibit mental retardation and developmental delay due to widespread demyelination; infant Krebbe patients often die by two years of age (14-16). Krabbe disease is the results of low GALC enzyme activity and a reduced ability to degrade galactolipids in myelin (13). Patients with Krabbe disease have decreased myelin due to the presence of psychosine, which is cytotoxic to oligodendroyctes, and the accumulation of galactosylceramide (13).
Similar to other Galc mouse models, the Crabby2 mice exhibit weight loss. Overt neurological phenotypes were not observed.
Crabby2(F):5'- TACTGCCTGAGTACACTGTACCCC -3'
Crabby2(R):5'- TTCACCAGTGACTACACATGCATCC -3'
Crabby2_seq(F):5'- CTGAGTACACTGTACCCCAATAG -3'
Crabby2_seq(R):5'- cttcttcttcttctccttcttcttc -3'
1. Deane, J. E., Graham, S. C., Kim, N. N., Stein, P. E., McNair, R., Cachon-Gonzalez, M. B., Cox, T. M., and Read, R. J. (2011) Insights into Krabbe Disease from Structures of Galactocerebrosidase. Proc Natl Acad Sci U S A. 108, 15169-15173.
2. Nagano, S., Yamada, T., Shinnoh, N., Furuya, H., Taniwaki, T., and Kira, J. (1998) Expression and Processing of Recombinant Human Galactosylceramidase. Clin Chim Acta. 276, 53-61.
3. Chen, Y. Q., Rafi, M. A., de Gala, G., and Wenger, D. A. (1993) Cloning and Expression of cDNA Encoding Human Galactocerebrosidase, the Enzyme Deficient in Globoid Cell Leukodystrophy. Hum Mol Genet. 2, 1841-1845.
4. Luzi, P., Rafi, M. A., Zaka, M., Curtis, M., Vanier, M. T., and Wenger, D. A. (2001) Generation of a Mouse with Low Galactocerebrosidase Activity by Gene Targeting: A New Model of Globoid Cell Leukodystrophy (Krabbe Disease). Mol Genet Metab. 73, 211-223.
5. Potter, G. B., Santos, M., Davisson, M. T., Rowitch, D. H., Marks, D. L., Bongarzone, E. R., and Petryniak, M. A. (2013) Missense Mutation in Mouse GALC Mimics Human Gene Defect and Offers New Insights into Krabbe Disease. Hum Mol Genet. 22, 3397-3414.
6. Sakai, N., Inui, K., Tatsumi, N., Fukushima, H., Nishigaki, T., Taniike, M., Nishimoto, J., Tsukamoto, H., Yanagihara, I., Ozono, K., and Okada, S. (1996) Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse, a Model of Krabbe's Disease. J Neurochem. 66, 1118-1124.
7. Lee, W. C., Tsoi, Y. K., Dickey, C. A., Delucia, M. W., Dickson, D. W., and Eckman, C. B. (2006) Suppression of Galactosylceramidase (GALC) Expression in the Twitcher Mouse Model of Globoid Cell Leukodystrophy (GLD) is Caused by Nonsense-Mediated mRNA Decay (NMD). Neurobiol Dis. 23, 273-280.
8. Duchen, L. W., Eicher, E. M., Jacobs, J. M., Scaravilli, F., and Teixeira, F. (1980) Hereditary Leucodystrophy in the Mouse: The New Mutant Twitcher. Brain. 103, 695-710.
9. Kobayashi, T., Yamanaka, T., Jacobs, J. M., Teixeira, F., and Suzuki, K. (1980) The Twitcher Mouse: An Enzymatically Authentic Model of Human Globoid Cell Leukodystrophy (Krabbe Disease). Brain Res. 202, 479-483.
10. Kodama, S., Igisu, H., Siegel, D. A., and Suzuki, K. (1982) Glycosylceramide Synthesis in the Developing Spinal Cord and Kidney of the Twitcher Mouse, an Enzymatically Authentic Model of Human Krabbe Disease. J Neurochem. 39, 1314-1318.
11. Visigalli, I., Ungari, S., Martino, S., Park, H., Cesani, M., Gentner, B., Sergi Sergi, L., Orlacchio, A., Naldini, L., and Biffi, A. (2010) The Galactocerebrosidase Enzyme Contributes to the Maintenance of a Functional Hematopoietic Stem Cell Niche. Blood. 116, 1857-1866.
12. Luzi, P., Rafi, M. A., and Wenger, D. A. (1995) Characterization of the Large Deletion in the GALC Gene found in Patients with Krabbe Disease. Hum Mol Genet. 4, 2335-2338.
13. Wenger, D. A., Rafi, M. A., Luzi, P., Datto, J., and Costantino-Ceccarini, E. (2000) Krabbe Disease: Genetic Aspects and Progress Toward Therapy. Mol Genet Metab. 70, 1-9.
14. De Gasperi, R., Gama Sosa, M. A., Sartorato, E. L., Battistini, S., MacFarlane, H., Gusella, J. F., Krivit, W., and Kolodny, E. H. (1996) Molecular Heterogeneity of Late-Onset Forms of Globoid-Cell Leukodystrophy. Am J Hum Genet. 59, 1233-1242.
15. Wenger, D. A., Rafi, M. A., and Luzi, P. (1997) Molecular Genetics of Krabbe Disease (Globoid Cell Leukodystrophy): Diagnostic and Clinical Implications. Hum Mutat. 10, 268-279.
|Science Writers||Anne Murray|
|Authors||Emre Turer and Bruce Beutler|
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