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Molecular Genetics of Krabbe

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              MUTATION UPDATE Molecular Genetics of Krabbe Disease (Globoid CellLeukodystrophy): Diagnostic and Clinical Implications David A. Wenger, *  Mohammad A. Rafi, and Paola Luzi Departments 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 thisdisease 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 GALCactivity 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; Contractgrant 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 samelow 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 subsequentlyfrom 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 and50- 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-                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           

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Jul 23, 2017
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