Adult onset globoid cell leukodystrophy (Krabbe disease): analysis of galactosylceramidase cDNA from four Japanese patients

Abstract

We examined galactosylceramidase (GALC) cDNA in four Japanese patients with adult onset globoid cell leukodystrophy (Krabbe disease; AO-GLD) by polymerase chain reaction/single-strand conformation polymorphism (PCR-SSCP) analysis, subsequent sequence determination, and restriction enzyme digestion of PCR products, initial symptoms were the onset of slowly progressive spastic paraplegia from the middle of the second decade, and all patients had diminished GALC activity in their leukocytes. We identified three missense mutations (I66M, G270D, L618S) and one exon-6 skipping (535-573del). Two of the patients had only the I66M mutant mRNA, and one only the G27OD mutant mRNA. The fourth patient carried a compound heterozygous mutation of 535-573del and L618S. To determine the enzymatic activities produced by these mutations, we constructed mutated GALC cDNAs and expressed them in COS-1 cells. Three mutations, viz., G270D, L618S, and exon-6 skipping (535-573del), produced diminished GALC activity as expected. The I66M mutation in the wild-type GALC cDNA(I289) had normal activity, but when this mutation and the V289 polymorphism were introduced into the same allele, it had decreased activity. Thus, the combination of a unique mutation and polymorphism causes conformational change in the GALC enzyme, resulting in low enzymatic activity. AO-GLD mutations, including those found here, are located in the N-terminus (I66M, G270D, 535-573del) or C-terminus (L618S) of the GALC enzyme, whereas the reported mutations in the infantile form (IF-GLD) are in the central domain. This difference in mutation sites may affect the clinical features of GLD.

Insights into Krabbe disease from structures of galactocerebrosidase

“…Mutations have been identified in the GALC gene that affect gene splicing and mRNA stability or cause deletions, frameshifts, and missense mutations (5,(13)(14)(15)(29)(30)(31)(32)(33)(34). A large proportion of the missense mutations in GALC that cause Krabbe disease are likely to result in protein mistargeting or premature degradation (33).…”

Newborn screening for Krabbe disease in New York State: the first eight years’ experience

“…Functional studies would be necessary to determine whether p.V320M and others (such as p.D94=) are indeed pseudodeficiency alleles. p.L618S was found in cis with several other variants in our population (Supplementary Table S3 online), and those who carried two copies of p.L618S were categorized as being at high (1; case 9, 3,28,29,32 Nearly all of the 348 infants referred carried at least one of the three common activity-attenuating polymorphisms (p.R168C, p.D232N, p.I546T). The frequency of each was elevated in the referral population compared with published data.…”

Six novel mutations detected in the GALC gene in 17 Japanese patients with Krabbe disease, and new genotype–phenotype correlation

“…Whereas several papers (Tatsumi et al 1995;Furuya et al 1997;Kukita et al 1997-98;Satoh et al 1997;Fu et al 1999) have reported Krabbe disease mutations in Japanese patients, clear genotype-phenotype correlations remain obscure, because of the small number of subjects studied. We evaluated the GALC gene in 17 Japanese patients, classifying mutations according to clinical phenotype.…”

“…1a). I66M+I289V, first reported by Furuya et al (1997), is a unique mutation identified in the Japanese population. Only when two single-nucleotide substitutions (I66M, I289V) resided on the same allele was their combination (I66M+I289V) proved to be a pathogenic mutation .…”