molecular genetics of krabbe

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MUTATION UPDATE

Molecular Genetics of Krabbe Disease (Globoid CellLeukodystrophy): Diagnostic and Clinical ImplicationsDavid A. Wenger, * Mohammad A. Rafi, and Paola LuziDepartments of Medicine (Medical Genetics) and Biochemistry and Molecular Pharmacology, Jefferson Medical College, Philadelphia,Pennsylvania

Communicated by Robert J. Desnick

Galactocerebrosidase (GALC) is a lysosomal -galactosidase responsible for the hydrolysis of the galac-tosyl 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 globoidcell leukodystrophy (GLD), because of the characteristic storage cells found around cerebral bloodvessels in the white matter of affected human patients and animal models. Although most patientspresent with clinical symptoms before 6 months of age, older patients, including adults, have beendiagnosed by their severe deficiency of GALC activity. More than 40 mutations have been identified inpatients with all clinical types of GLD. While some mutations clearly result in the infantile type if foundhomozygous or with another severe mutation, it is difficult to predict the phenotype of novel mutationsor when mutations are found in the heterozygous state. A high incidence of polymorphic changes onapparent disease-causing alleles also complicates the interpretation of the effects of mutations. Thedetection of mutations has greatly improved carrier identification among family members and will per-mit preimplantation diagnosis for some families. The molecular characterization of the naturally occur-ring mouse, dog, and monkey models will permit their use in trials to evaluate different modes of therapy. Hum Mutat 10:268–279, 1997. © 1997 Wiley-Liss, Inc.

KEY WORDS : GALC; GLD; preimplantation diagnosis

Krabbe disease, or globoid cell leukodystrophy(GLD, MIM 24520), is an autosomal recessive disor-der resulting from the deficiency of galactocere-brosidase (GALC, EC 3.2.1.46) activity (for review,see Wenger, 1997; Suzuki et al., 1995). The incidenceof the most common infantile form is estimated to be1:100,000 in the United States and Northern Eu-rope. There is an extremely high frequency in someinbred communities such as the Druze and someMoslem Arab villages in Israel (Zlotogora et al., 1985,1991). The clinical stages of the infantile form of this

disease were clearly described by Hagberg and col-leagues (1970). Most patients present before sixmonths of age with irritability, spasticity, arrest of motor and mental development, and bouts of tem-perature elevation without infection. This is followedby myoclonic jerks of arms and legs, oposthotonus,hypertonic fits, and mental regression, whichprogresses to a severe decerebrate condition with novoluntary movements and death from respiratoryinfections or cerebral hyperpyrexia before 2 years of

age. Such cases make up about 90% of the nearly300 cases diagnosed in this laboratory alone. How-ever, a significant number of cases with later onset,presenting with unexplained blindness, weakness and/ or progressive motor, and sensory neuropathy thatcan progress to severe mental incapacity and death,have been identified (Crome et al., 1973; Thomas etal., 1984; Verdu et al., 1991; Kolodny et al., 1991;Wenger, 1997; Luzi et al., 1996).

The deficiency of GALC was identified as thecause of GLD by Malone (1970) and Suzuki andSuzuki (1970). Patients with GLD of any age are de-finitively diagnosed by their extremely low GALC

activity measured in any available tissue sample, in-cluding cultured amniotic fluid cells (Suzuki andSuzuki, 1971; Suzuki et al., 1971; Wenger et al.,1974). This laboratory alone has accurately per-

Received 4 October 1996; accepted 19 January 1997.Contract grant sponsor: National Institutes of Health; Contract

grant number: DK38795.*Correspondence to: David A. Wenger, Departments of Medicine

(Medical Genetics) and Biochemistry and Molecular Pharmacology, Jefferson Medical College, Philadelphia, PA 19107; Fax: 215-955-9554.

HUMU 833D



formed more than 300 prenatal tests for Krabbe dis-ease by measuring GALC activity in a sample of chori-onic villus obtained at about 9–10 weeks gestation.The residual GALC activity measured in the diag-nostic assay is not directly correlated to the clinicalcourse, and many later-onset patients have the same

low level of enzymatic activity as the infantile pa-tients (Young et al., 1972; Wenger et al., 1974; Vanier,1991; Kolodny et al., 1991; Luzi et al., 1996). In fact,some healthy individuals have been found to havequite low GALC activity (Wenger and Riccardi,1976). This lack of correlation between the in vitroresidual activity and the onset of disease probablyreflects at least four considerations. Although radio-labeled natural substrate, galactosylceramide, is usedin the assay in this laboratory, the detergents used tostimulate substrate–enzyme interaction are not usedin lysosomes. In vivo this enzyme may require saposinC (SAP-2) and phosphatidylserine for the lysosomalhydrolysis of galactosylceramide (Wenger et al.,1982). While GALC catalyzes the hydrolysis of galactosylceramide, the inability to hydrolyze anothernatural substrate, galactosylsphingosine (psychosine),may, in fact, be the cause of the pathology and symp-toms observed in this disease (Svennerholm et al.,1980; Igisu and Suzuki, 1984; Kobayashi et al., 1987).Some mutations in this gene may affect the ability of GALC to hydrolyze psychosine differently thangalactosylceramide. While easily obtained tissuesamples are used for pre- and postnatal diagnosis, theactivities measured may not reflect the residual ac-tivities in the central and peripheral nervous systems.Finally, GALC is an extremely hydrophobic lysosomalenzyme, existing as a very large complex composed of 30-kD and 50- to 52-kD subunits derived from the 80-kD precursor (Chen and Wenger, 1993). With muta-tions occurring in the coding regions of each subunitand some consisting of large deletions, it is difficult topredict the consequences of such mutations on GALCactivity. Also, in some patients an unequal number of “normal” subunits may disrupt the formation of thehigh-molecular-weight complex.

The first purification of this extremely low abun-dant enzyme was from human urine and subsequently

from human brain (Chen and Wenger, 1993). Onlyenough purified protein was available for N-terminalamino acid sequencing of subunits separated by so-dium dodecyl sulfate–polyacrylamide gel electro-phoresis (SDS-PAGE). Antibodies prepared from lessthan pure fractions were not specific enough to usein screening an expression library, or to use in bio-synthesis and processing experiments or for measure-ment of antigen levels. Antibodies against the 12N-terminal amino acids of the 80-kD precursor and

50- to 52-kD subunits conjugated to bovine serum al-bumin and keyhole limpet hemocyanin were prepared.They will only detect partially purified 80-kD and 50-to 52-kD subunits on Western blotting after SDS-PAGE. Sakai and coworkers (1994a) later describedthe purification of GALC from human lymphocytes.

Krabbe disease was mapped to human chromo-some 14 by linkage analysis using a number of thelarger families available to us (Zlotogora et al., 1990).Identification of patients and carriers was done bymeasurement of GALC activity in leukocytes. Diffi-culties in accurate carrier identification by thismethod became more evident when these large fami-lies were studied. It became obvious that there areseveral “normal” alleles responsible for the wide rangeof GALC values measured in vitro. However, sincethe contribution of each allele appears to be addi-tive, the availability of parents made carrier predic-tion quite reliable in their offspring (Zlotogora et al.,1990; Wenger, 1997). Eventually Krabbe disease andthe GALC gene were mapped to the region 14q25–31 by the use of CA repeat polymorphisms (Oehl-mann et al., 1993) and in situ hybridization(Cannizzaro et al., 1994).

Using the amino acid sequence from the N-ter-minals of the 50- to 52-kD subunits and the 80-kDprecursor, degenerate primers were synthesized, anda PCR product was amplified using RNA from cattestes. When this amplified fragment was used toscreen a human kidney library, the full-length cDNAsequence was obtained (Chen et al., 1993; GenBankAccession No. L23116). The cDNA is about 3.8-kbin length, including 47 nt of 5' untranslated region,2007 nt of open reading frame(ORF) (coding for 669amino acids), and 1741 nt of 3' untranslated sequence(Chen et al., 1993). The cDNA has no significanthomology to any sequence in the data bank. North-ern blot of polyA+ RNA only detected the 3.8-kbspecies indicating the use of the most 3' polyaden-

ylation signal. However, due to the presence of a cryp-tic polyadenylation signal at the 5' end of intron 10,a shortened mRNA species could be detected in pa-

tients and carriers who have a large deletion begin-ning near the middle of intron 10 (see below).The sequence around the initiation codon is atypi-

cal in the +4 position having an A instead of themore common G (Kozak, 1989). This could be onecause for the low expression of this gene. It is ex-tremely difficult to detect GALC mRNA on North-ern blots of total RNA from controls. The first 78 ntof the ORF code for a 26-amino acid leader peptide.The remaining 643 amino acids contain six poten-



tial glycosylation sites, three in each subunit. Twosites in the 30-kD subunit are only 2 amino acidsapart and probably are not both used. Subsequently,the full-length human GALC cDNA was also clonedfrom human placenta (Sakai et al., 1994b; GenBankAccession Nos. D25283 and D25284)

The human GALC gene is about 58 kb in lengthconsisting of 17 exons and 16 introns (Luzi et al.,1995a; Accession Nos. L38544 to L38559). The ex-ons range in size from 39 to 181 bp, and the intronsrange in size from 247 bp (intron 2) to about 12 kb(intron 10). Intron 10 contains a stretch of at least17 CA repeats beginning at nucleotide 762, and apolyadenylation signal beginning at nucleotide 51,both counting from the start of intron 10. Thispolyadenylation signal is used in those genes thatcontain certain mutations, including the large dele-tion beginning near the middle of intron 10 (Rafi etal., 1995). The promoter region consists of GC box-type consensus sequences 5' to the start site andwithin intron 1. Promoter activity was detected inthe 290 bp preceding the start site (Luzi P, Rafi MA,and Wenger DA, unpublished observations). Thesefinding are consistent with its role as a housekeepinggene with an extremely low level of expression.

Using a variety of techniques, mutations wereidentified in many patients available to this labora-tory. A significant number of the mutations presentedon Table 1 have not been previously published. If RNA from cultured cells was available, it was sub-jected to reversed transcribed polymerase chain re-action (RT-PCR), followed by direct sequencing of the PCR products. Once the intronic sequences be-came available, all exons and exon–intron bound-aries were amplified using intronic primers andsequenced directly. All mutations identified as ho-mozygous by RT-PCR were confirmed using genomicDNA to rule out the possibility of low GALC mRNAin one mutated allele. Over 80 patients have beenpartially or completely characterized in this labora-tory alone, and a number of mutations have beenreported (Rafi et al., 1995, 1996a; Luzi et al., 1995b,c,1996). Additional mutations have been identified byother groups (Sakai et al., 1994b; Kolodny et al., 1995;Tatsumi et al., 1995). Mutation analysis has resultedin the identification of over 40 changes from the mostcommon sequence. While most of these changes areconsidered disease-causing because they occur aloneand /or produce very low GALC activity in transientexpression studies, a number of these alterations areconsidered normal polymorphisms. Some missensemutations are found on the same allele as an obvi-ously severe mutation (deletion, insertion, nonsense

mutation). The possible consequences of inheritingmultiple polymorphic changes in this gene will bediscussed below. However as shown in Table 1, moreoften than not, disease-causing mutations will befound together with a polymorphic change on thesame allele. The most commonly found polymor-

phisms include a C>T change at cDNA position 502(R169C), a G>A at position 694 (D232N), and T>Cat position 1637 (I546T). Many patients have mul-tiple changes in one or both alleles, and many arecompound heterozygotes. This will, in some cases,prevent assigning a phenotype to a mutation. Exceptwhere indicated, all mutations are named using thecDNA sequence number counting from the A of theinitiation codon and the nature of the mutation.Mutations are listed in Table 1; their locations onthe gene are shown in Figure 1. First, we discuss thosemutations found in the homozygous state.

We have identified one mutation present in about40% of the chromosomes of patients with Krabbedisease of European ancestry (Rafi et al., 1995; Luziet al., 1995b). When this mutation is found in thehomozygous state or together with a severe muta-tion in the second allele, it results in the infantileform of Krabbe disease. It has also been found in theheterozygous state in five juvenile and adult patients.This mutation, called 502T/del, consists of a 30-kbdeletion which starts near the middle of the 12-kbintron 10 (at intron nucleotide number 6532) andcontinues about 9 kb beyond the polyadenylation sig-nal. This mutation eliminates all of the coding re-gion for the 30-kD subunit and 15% of the codingregion for the 50-kD subunit; no activity is producedby expression studies (Rafi et al., 1995). Howeverdue to the presence of a polyadenylation signal nearthe 5' end of intron 10, a normal amount of shortGALC mRNA is seen in individuals heterozygous orhomozygous for this mutation. This deletion is al-ways found on the allele containing the C>T changeat cDNA position 502, where T is usually found inonly 5% of the chromosomes in the general popula-tion. The C>T change is at a CpG dinucleotide. Itappears that the large deletion occurred once on thebackground of the 502T allele, since all alleles withthe deletion have 502T. But not every allele with502T has the 30-kb deletion. Although the exactmechanism for the deletion is not known, the dele-tion junction contains a four nucleotide direct re-peat (Luzi et al., 1995b). Simple tests for the genotypeat 502 and the deletion have been developed (Rafiet al., 1995; Luzi et al., 1995b). From the ancestry of the patients diagnosed with this deletion, one can con-clude that it must have originated in Sweden many

years ago and have spread throughout Northern Eu-rope and possibly the Iberian peninsula, eventually



(D171V), G>C at 533 (G178A), G>A at 739(A247T), G>A at 802 (G268S), G>A at 809(G270D), A>C at 836 (N279T), C>G at 904(P302A), C>T at 1151 (P384L), C>G at 1355(T452S), T>C at 1493 (F498S), C>T at 1543(R515C), G>T at 1726 (A576S), and G>A at 1873

(A625T). In some cases the second allele was one of the more common alleles, such as 502T/del orC1538T, and in others it was unique (Table 1). Insome patients the second mutated allele has not beenidentified due to a lack of sample.

Two nonsense mutations have been found. The firstmutation identified was a G>T transversion at 1105(E369X) in an infantile non-Japanese patient (Sakai etal., 1994b). The most 5' mutation resulting in a stopcodon in the signal peptide (C>A at position 20 lead-ing to S7X) was found in a 35-year-old patient who hasretained normal intellect in the presence of severe motorinvolvement. That allele also contains two polymor-phisms, 502T and 1637C. The other allele in that pa-tient contains A>T at 301 (M101L) and 1637C.

Six single base deletions and one single base in-sertion have been identified, 382delA, 805delG,906delT, 964delG, 1424delA, 1675insT, and1852delT (Table 1). Six of these mutations result ina frameshift leading to a premature stop codon. Onedeletion in a Mexican patient (1852delT) resultedin a stop codon (L618X). It is interesting that threeof these individuals had another missense mutationon the same allele. The individual with the 382delAmutation also had G>T at 1726 (A576S) on thesame allele. The 1424delA mutation was found onthe same allele with G>A at 1873 (A625T), and1675insT was found on the same allele with C>G at1355 (T452S). It is important to note that Kolodnyet al. (1995) found the G>A change at 1873 in alate-onset patient, but did not report the 1424delAmutation we found on the same allele. It would becritical to know whether the deletion of A at 1424was present in the patient of Kolodny and cowork-ers. Most of these single nucleotide deletions andinsertions had one or more polymorphic changes onthe same allele including one with both 502T and1637C, three with 1637C and one with 694A (Table

1). One allele with a single base pair deletion had noadditional changes in the coding region, and onepatient was not studied further.

Other than the 502T/del (Rafi et al., 1995) andthe 635del+ins (Tatsumi et al., 1995) mutationsmentioned earlier, additional deletions of more thanone nucleotide were found. We have found that oneallele of an infantile Italian patient had a deletion of 10 nts within exon 10 (1026del10). This leads to aframeshift and premature stop codon. That patient

was heterozygous for the A>C change at 1652 foundin other European patients. One infantile patient whowas heterozygous for the 502T/del allele was foundto have a large deletion (about 12 kb) beginning nearthe 5' end of intron 10 (at intronic position 262) andincluding the first 26 nt of exon 11 (Int10del). This

large deletion may also use the cryptic polyadenylation signal located near the 5' end of intron 10.

One infantile patient of Mexican ancestry wasfound to have an unusual genotype. She was het-erozygous for the 502T/del allele plus a complex al-lele. The last nucleotide of intron 6 was changed fromg to t (574-1g>t), which resulted in the loss of thesplice acceptor site. Alternative splicing lead to thesplicing out of the first 8 nts of exon 7. This resultedin a frameshift and stop codon in that exon. In addi-tion, that allele also contained a deletion of 17 nt atposition 1822 in exon 16, which also lead to a frame-shift and stop codon after 7 amino acids. That allelealso had the polymorphic T>C change at position 1637.

It has been known for many years that identifica-tion of carriers of Krabbe disease based on measure-ment of GALC activity was very difficult (Svennerholmet al., 1981; Mansson and Svennerholm, 1982; Wenger,1997). This was partially due to the wide range of “nor-mal” values measured in white blood cells and culturedskin fibroblasts. However, due to the additive nature of the contribution from each allele, carriers usually couldbe identified if GALC values from the parents wereavailable (Zlotogora et al., 1990). It was proposed thatthis variability probably resulted from the presence of polymorphic changes in the GALC gene. After clon-ing the GALC cDNA (Chen et al., 1993), we startedsequencing those patients where RNA could be ob-tained. It became evident early in our search for muta-tions that the common 30-kb deletion was always foundtogether with a 502 C>T change (R168C). Expres-sion of 502T as the only change in the cDNA sequencein COS-1 cells resulted in about 10–20% less GALCactivity than the most common sequence (Rafi et al.,1995; Luzi et al., 1996).

Additional sequencing of other patients lead tothe identification of the T>C change at position1637. This also was identified as a polymorphism bySakai et al. (1994b). COS-1 cell expression of 1637Cas the only change, resulted in the measurement of only about 30% as much GALC activity as the allelewith 1637T (Luzi et al., 1996). In fact, this polymor-phism is the major cause for the wide range of “nor-mal” GALC activity. Its presence adds to theconfusion of interpreting GALC values if found in



reading frame is 2007, identical to human and dog,and the nucleotide sequence is nearly 98% identicalto human. No nucleotide changes were found on se-quencing the mother’s GALC mRNA by RT-PCR,indicating that the mutant allele may make a verylow level of GALC mRNA. A breeding of this fe-

male with her healthy son, recently resulted in thebirth of an affected monkey (Baskin GB, RatterreeM, Davison BB, Falkenstein KP, Clarke MR, England

JD, Vanier MT, Luzi P, Rafi MA, Wenger DA, sub-mitted). Using primers generated from the sequenceof the human GALC gene (Luzi et al., 1995a) allexons and exon–intron boundaries of the affectedmonkey’s DNA were sequenced. A deletion of twonts in exon 4 was found, and this results in a frame-shift and premature stop codon (Luzi et al., 1997).This probably results in unstable mRNA and explainsthe low level of mutant GALC mRNA in the het-erozygous mother. A rapid method for detecting thismutation has been developed, and all members of this colony will be checked for carrier status. As thisis the only nonhuman primate model of a leukodys-trophy it will provide unique opportunities to evalu-ate several therapeutic modalities, including in uteroand neonatal bone marrow transplantation and genetherapy using transduced hematopoietic stem cells.

The purification of GALC and the cloning of itscDNA and gene has improved our diagnostic capa-bilities as well as provide opportunities for future at-tempts at therapy, including gene therapy. Theidentification of disease-causing mutations in a pa-tient immediately opens the door to accurate carriertesting for interested family members. It also providesan opportunity for preimplantation diagnosis andimplantation of only unaffected embryos. While not

yet tried for couples at risk of having a child withKrabbe disease, it has been done for other autoso-mally inherited disorders (Handyside et al., 1992;Gibbons et al., 1995). It has been difficult to makeclear genotype-phenotype correlations when patientsare heterozygous for two mutations, except when bothmutations result in severely defective enzyme. Inter-

preting the clinical consequences of inheriting com-plex alleles is very difficult (Scott et al., 1993; Kappleret al., 1994). Since some individuals are heterozy-gous for a severe mutation, such as 502T/del, andanother novel mutation, it usually is not possible topredict the clinical outcome. It is possible that some“mild” mutations only produce disease when the sec-ond allele has a severe mutation. This may be truefor some mutations found in adult patients. Whilesome investigators have successfully correlated the

phenotypes with studies of mutations, residual enzy-matic activities, and antigen levels in some diseases(Litjens et al., 1996), in reality this information addslittle to predicting the clinical course in a newly di-agnosed patient. Careful clinical evaluation of thepatient will most likely predict the clinical course

within some boundaries that can obviously be modi-fied by nongenetic events such as infections and feed-ing difficulties. At this time, the lack of suitableantibodies against human GALC prevent furtherstudies looking at antigen levels and biosynthesis andprocessing in cultured cells.

The possible clinical consequences of inheritingthree or more polymorphic mutations in the GALCgene have been considered (Luzi et al., 1995c). Inthis lysosomal diseases testing laboratory, almost allpatients of any age who are experiencing neurologi-cal deterioration are tested for GALC activity. Wehave noticed that a number of individuals with clini-cal and test evidence, such as magnetic resonanceimaging (MRI), of white matter disease have GALCactivity 10-25% of our normal mean. These valuesare clearly above those found in individuals with alltypical forms of GLD, including adult onset. WhenDNA from these individuals was examined for theirgenotypes at the polymorphic C502T and T1637Cpositions, a highly significant number of these peoplewere found to have three or four copies of the leastcommon nucleotides at these two positions. Someindividuals were homozygous for both 502T and1637C. As these patients have a wide range of clini-cal features and age of onset, it is difficult to see howthe presence of these mutations are solely respon-sible for the unexplained white matter degeneration.It is possible that inheriting multiple copies of poly-morphic changes in the GALC gene is a predispos-ing factor responsible for white matter disease in asignificant number of undiagnosed individuals. Fu-ture studies using transgenic mice with similar aminoacid changes will determine whether the presence of lower than normal, but not totally deficient, GALCactivity has an effect on the structure of myelin andability of such myelin to respond to demyelinatinginsults and remyelinate upon removal of the insult.

While GALC was difficult to purify because of itslow abundance and extreme hydrophobicity, it ispossible that these factors will be useful as we con-sider treating animals and humans with this disease.If a very low level of this enzyme is sufficient to pre-vent disease, only a very low level may be required totreat the cells needing it. Also, while the enzyme isextremely hydrophobic and aggregates into a highmolecular weight complex, it also may be taken upby neighboring cells by non-receptor-mediated



mechanisms. Two recent studies have demonstratedretroviral gene correction of cells from GLD patients(Gama Sosa et al., 1996; Rafi et al., 1996b). Our stud-ies using human GALC cDNA in a retroviral vector(MFG-GALC) have demonstrated that overexpres-sion of GALC activity (100–200 times normal) in

cultured skin fibroblasts is not toxic to cells 4 monthsafter transduction and many doublings. These cellssecrete GALC into the medium that is rapidly takenup by neighboring cells. Deficient untransduced cellscan attain normal GALC levels after 2 days in con-tact with the medium containing GALC activity. Thisuptake of secreted enzyme by cells is inhibited lessthan 50% by mannose-6-phosphate indicating thatthe transfer is not solely via the mannose-6-phos-phate receptor-mediated pathway. In addition tocultured fibroblasts, successful transduction of myo-blasts, rat brain astrocytes, mouse Schwann cells, andhuman and canine hematopoietic cells has been dem-onstrated (Rafi et al., 1996b; Rafi MA, Luzi P, andWenger DA, unpublished observations). Treatmentof the human patients has been limited to bone mar-row transplantation, and the results indicate thattransplantation before serious neurological compli-cations occur slows the progression of the disease(Shapiro et al., 1991). Studies on the mouse, dog,and monkey models are in progress, and they shouldprovide information critical to the successful treat-ment of the human patients.

This research was supported in part by grantDK38795 from the National Institutes of Health. Theauthors thank Teresa Victoria, Caren Dubell, MaryamShahinfar, Gregory de Gala, H. Zhi Rao, and H. XianShen for their excellent help on this project.

Mutation 805delG reported by Kolodny et al.(1995) was shown by Rafi MA, Luzi P, and WengerDA to be an error, and that the patient had theG809A mutation seen in a number of other juvenileand adult patients. This was corrected in an Erratumpublished in Am J Hum Genet 60:1264 (1997).

Baskin G, Alroy J, Li Y-T, Dayal Y, Raghavan SS, Sharer L (1989)Galactosylceramide-lipidosis in Rhesus monkeys. Lab Invest 60:7A.

Cannizzaro LA, Chen YQ, Rafi MA, Wenger DA (1994) Regionalmapping of human galactocerebrosidase (GALC) to 14q31 by insitu hybridization. Cytogenet Cell Genet 66:244–245.

Chen YQ, Wenger DA (1993) Galactocerebrosidase from humanurine: Purification and partial characterization. Biochim BiophysActa 1170:53–61.

Chen YQ, Rafi MA, de Gala, G, Wenger DA (1993) Cloning andexpression of cDNA encoding human galactocerebrosidase, the

enzyme deficient in globoid cell leukodystrophy. Hum Mol Genet2:1841–1845.

Crome L, Hanefield F, Patrick D, Wilson J (1973) Late onset globoidcell leucodystrophy. Brain 96:841–848.

Fankhauser R, Luginbuhl H, Hartley WJ (1963) Leukodystrophie vomtypus Krabbe beim hund. Schweiz Arch Tierheilkd 105:198–207.

Fletcher TF, Kurtz HJ, Low DG (1966) Globoid cell leukodystrophy

(Krabbe type) in the dog. J Am Vet Med Assoc 149:165–172.Gama Sosa MA, De Gasperi R, Undevia S, Yeretsian J, Rouse II SC,

Lyerla TA, Kolodny EH (1996) Correction of the galacto-cerebrosidase deficiency in globoid cell leukodystrophy-culturedcells by SL-3 retroviral-mediated gene t ransfer. Biochem BiophysRes Commun 218:766–771.

Gibbons WE, Gitlin SA, Lanzendorf SE, Kaufmann RA, SlotnickRN, Hodgen GD (1995) Preimplantation genetic diagnosis forTay-Sachs disease:successful pregnancy after pre-embryo biopsyand gene amplification by polymerase chain reaction. Fertil Steril63:723–728.

Hagberg B, Kollberg H, Sourander P, Akesson HO (1970) Infantilegloboid cell leucodystrophy (Krabbe’s disease): A clinical andgenetic study of 32 Swedish cases 1953–1967. Neuropaediatrie1:74–88.

Handyside AH, Lesko JG, Tarin JJ, Winston RML, Hughes MR (1992) Birth of a normal girl after in vitro fertilization and pre-implantation diagnosis testing for cystic fibrosis. N Engl J Med327:905–909.

Hoogerbrugge PM, Suzuki K, Suzuki K, Poorthuis BJHM, KobayashiT, Wagemaker G, van Bekkum DW (1988a) Donor-derived cellsin the central nervous system of twitcher mice afte r bone marrowtransplantation. Science 239:1035–1038.

Hoogerbrugge PM, Poorthuis BJ, Romme AE , van de Kamp JJ,Wagemaker G, van Bekkum DW (1988b) Ef fect of bone ma r-row transplantation on enzyme levels and clinical course inthe neurologically affected twitcher mouse. J Clin Invest81:1790–1794.

Ichioka T, Kishimoto Y, Brennan S, Santos GW, Yeager AM (1987)Hematopoietic cell transplantation in murine globoid cell leu-kodystrophy (the twitcher mouse): Effects on levels of galactosylceramidase, psychosine, and galactocerebrosides. ProcNatl Acad Sci USA 84:4259–4263.

Igisu H, Suzuki K (1984) Progressive accumulation of toxic metabo-lite in a genetic leukodystrophy. Science 224:753–755.

Kappler J, Sommerlade HJ, von Figura K, Gieselmann V (1994) Com-plex arylsulfatase A alleles causing metachromatic leukodystro-phy. Hum Mutat 4:119–127.

Kobayashi T, Yamanaka T, Jacobs J, Teixeira F, Suzuki K (1980) Thetwitcher mouse: An enzymatically authentic murine model of human globoid cell leukodystrophy (Krabbe disease). Brain Res202:479–483.

Kobayashi T, Shinoda H, Goto I, Yamanaka T, Suzuki Y (1987)Globoid cell leukodystrophy is a generalized galactosylsphingosine(psychosine) storage disease. Biochem Biophys Res Commun144:41–46.

Kolodny EH, Raghavan S, Krivit W (1991) Late-onset Krabbe dis-

ease (globoid cell leukodystrophy): clinical and biochemical fea-tures of 15 cases. Dev Neurosci 13:232–239.Kolodny EH, Gama Sosa MA, Battistini S, Sartorato EL, Yeretsian J,

MacFarlane H, Gusella J, De Gasperi R (1995) Molecular genet-ics of late-onset Krabbe disease. Am J Hum Genet 57:A217.

Kozak M (1989) The scanning model for translation: an update. JCell Biol 108:229–241.

Litjens T, Brooks DA, Peters C, Gibson GJ, Hopwood JJ (1996) Iden-tification, expression, and biochemical characterization of N-acetylgalactosamine-4-sulfatase mutations and relationship withclinical phenotype in MPS-VI patients. Am J Hum Genet58:1127–1134.



Luzi P, Rafi MA, Wenger DA (1995a) Structure and organization of the human galactocerebrosidase (GALC) gene. Genomics26:407–409.

Luzi P, Rafi MA, Wenger DA (1995b) Characterization of the largedeletion in the GALC gene found in patients with Krabbe dis-ease. Hum Mol Genet 4:2335–2338.

Luzi P, Rafi MA, Wenger DA (1995c) Krabbe disease: Correlationsbetween mutations in the GALC gene, GALC activity and clini-cal presentation. Am J Hum Genet 57:A245.

Luzi P, Rafi MA, Wenger DA (1996) Multiple mutations in the GALCgene in a patient with adult-onset Krabbe disease. Ann Neurol40:116–119.

Luzi P, Rafi MA, Victoria T, Baskin GB, Wenger DA (1997) Charac-terization of the rhesus monkey galactocerebrosidase (GALC)cDNA and gene, and identification of the mutation causinggloboid cell leukodystrophy (Krabbe disease) in this primate.Genomics 42:319–324.

Malone M (1970) Deficiency in degradative enzyme system in globoidleucodystrophy. Trans Am Soc Neurochem 1:56.

Mansson J-E, Svennerholm L (1982) The use of galactosylceramideswith uniform fatty acids as substrates in the diagnosis and carrierdetection of Krabbe disease. Clin Chim Acta 126:127–133.

Naidu S, Hofmann KJ, Moser HW, Maumenee IH, Wenger DA (1988)Galactosylceramide â-galactosidase deficiency in association withcherry red spot. Neuropediatrics 19:46–48.

Oehlmann R, Zlotogora J, Wenger DA, Knowlton RG (1993) Local-ization of Krabbe disease gene (GALC) on chromosome 14 bymultipoint linkage analysis. Am J Hum Genet 53:1250–1255.

Rafi MA, Luzi P, Chen YQ, Wenger DA (1995) A large deletion to-gether with a point mutation in the GALC gene is a commonmutant allele in patients with infantile Krabbe disease. Hum MolGenet 4:1285–1289.

Rafi MA, Luzi P, Zlotogora J, Wenger DA (1996a) Two different mu-tations are responsible for Krabbe disease in the Druze and Mos-lem Arab populations in Israel. Hum Genet 97:304–308.

Rafi MA, Fugaro J, Amini S, Luzi P, de Gala G, Victoria T, Dubell C,Shahinfar M, Wenger DA (1996b) Retroviral vector-mediatedtransfer of the galactocerebrosidase (GALC) cDNA leads tooverexpression and transfer of GALC activity to neighboring cells.Biochem Mol Med 58:142–150.

Sakai N, Inui K, Midorikawa M, Okuno Y, Ueda S, Iwamatsu A,Okada S (1994a) Purification and characterization of galactocere-brosidase from human lymphocytes. J Biochem 116:615–620.

Sakai N, Inui K, Fujii N, Fukushima H, Nishimoto J, Yanagihara I,Isegawa Y, Iwamatsu A, Okada S (1994b) Krabbe disease:Isolationand characterization of a full-length cDNA for human galac-tocerebrosidase. Biochem Biophys Res Commun 198:485–491.

Sakai N, Inui K, Tatsumi N, Fukushima H, Nishigaki T, Taniike M,Nishimoto J, Tsukamoto H, Yanagihara, Ozono K, Okada S (1996)Molecular cloning and expression of cDNA for murinegalactocerebrosidase and mutation analysis of the twitcher mouse,a model of Krabbe’s disease. J Neurochem 66:1118–1124.

Scott HS, Nelson PV, Litjens T, Hopwood JJ, Morris CP (1993) Mul-tiple polymorphisms within the á-L-iduronidase gene (IDUA):

Implications for a role in modification of MPS-I disease pheno-type. Hum Mol Genet 2:1471–1473.Shapiro E, Lockman L, Kennedy W, Zimmerman D, Kolodny E,

Raghavan S, Wiederschain G, Wenger DA, Sung JH, SummersCG, Krivit W (1991) Bone marrow transplantation as treatmentfor globoid cell leukodystrophy. In Desnick R (ed): Treatment of Genetic Diseases. New York: Churchill Livingstone, pp 223–238.

Suzuki K, Suzuki Y (1970) Globoid cell leucodystrophy (Krabbe’sdisease): Deficiency of galactocerebroside â-galactosidase. ProcNatl Acad Sci USA 66:302–309.

Suzuki Y, Suzuki K (1971) Krabbe’s globoid cell leukodystrophy:

Deficiency of galactocerebrosidase in serum, leukocytes, and fi-broblasts. Science 171:73–75.

Suzuki K, Austin J, Armstrong D, Suzuki K, Schlenker J, Fletcher T(1970) Studies in globoid leukodystrophy: Enzymatic and lipidfindings in the canine form. Exp Neurol 29:65–75.

Suzuki K, Suzuki K (1985) Genetic galactosylceramidase deficiency(globoid cell leukodystrophy, Krabbe disease) in different mam-malian species. Neurochem Pathol 3:53–68.

Suzuki K, Schneider EL, Epstein CJ (1971) In utero diagnosis of globoid cell leukodystrophy (Krabbe’s disease). Biochem BiophysRes Commun 45:1363–1366.

Suzuki K, Suzuki Y, Suzuki K (1995) Galactosylceramide lipidosis:Globoid cell leukodystrophy (Krabbe disease). In Scriver CR,Beaudet AL, Sly WS, Valle D (eds): The Metabolic and Molecu-lar Bases of Inherited Disease. 7th Ed. New York: McGraw-Hill,pp 2671–2692.

Svennerholm L, Vanier M-T, Mansson J-E (1980) Krabbe disease: Agalactosylsphingosine (psychosine) lipidosis. J Lipid Res 21:53–64.

Svennerholm L, Vanier M-T, Hakansson G, Mannson J-E (1981) Useof leukocytes in diagnosis of Krabbe disease and detection of car-riers. Clin Chim Acta 112:333–342.

Tatsumi N, Inui K, Sakai N, Fukushima H, Nishimoto J, Yanagihara I,Nishigaki T, Tsukamoto H, Fu L, Taniiki M, Okada S (1995)Molecular defects in Krabbe disease. Hum Mol Genet 4:1865–1868.

Thomas PK, Halpern J-P, King RHM, Patrick P (1984) Galactosyl-ceramide lipidosis: Novel presentation as a slowly progressivespinocerebellar degeneration. Ann Neurol 16:618–620.

Vanier M (1991) Symptomatology of late onset Krabbe’s leukod- yst rophy: The European experience. Dev Neurosc i 13:240–244.

Verdu P, Lammens M, Dom R, Van Elsen A, Carton H (1991) Globoidcell leukodystrophy: a family with both late-infantile and adulttype. Neurology 41:1382–1384.

Victoria T, Rafi MA, Wenger DA (1996) Cloning of the canine GALCcDNA and identification of the mutation causing globoid cellleukodystrophy in West Highland White and Cairn terriers.Genomics 33:457–462.

Wenger DA (1997) Krabbe disease (globo id cell leukodystrophy).In Rosenberg RN, Prusiner SB, DiMauro S, Barchi RL (eds):The Molecular and Genetic Basis of Neurological Disease.Boston: Butterworth–Heinemann, pp 421–431.

Wenger DA, Riccardi VM (1976) Possible misdiagnosis of Krabbedisease. J Pediatr 88:76–79.

Wenger DA, Sattler M, Clark C, McKelvey H (1974) An improvedmethod for the identification of patients and carriers of Krabbe’sdisease. Clin Chim Acta 56:199–206.

Wenger DA, Sattler M, Roth S (1982) A protein activator of galactosylceramide â-galactosidase. Biochim Biophys Acta712:639–649.

Yeager AM, Brennan S, Tiffany C, Moser HW, Santos GW (1984)Prolonged survival and remyelination after hematopoietic celltransplantation in the twitcher mouse. Science 225:1053–1054.

Young E, Wilson J, Patrick AD, Crome L (1972) Galactocerebosidasedeficiency in globoid cell leucodystrophy of late onset. Arch DisChild 47:449–450.

Zlotogora J, Regev R, Zeigler M, Iancu TC, Bach G (1985) Krabbedisease: Increased incidence in a highly inbred community. Am JMed Genet 27:765–770.

Zlotogora J, Chakraborty S, Knowlton RG, Wenger DA (1990) Krabbedisease locus mapped to chromosome 14 by genetic linkage. Am

J Hum Genet 47:37–44.Zlotogora J, Levy-Lahad E, Legum C, Iancu TC, Zeigler M, Bach G

(1991) Krabbe disease in Israel. Isr J Med Sci 27:196–198.

molecular genetics of krabbe

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