Krabbe disease is an autosomal recessive leukodystrophy caused by the deficiency of the galactocerebrosidase (GALC) enzyme. It is pathologically characterized by demyelination of the central and peripheral nervous systems by accumulation of galactosylsphingosine. To date, more than 120 mutations in the GALC gene have been reported worldwide and genotype-phenotype correlations have been reported in some types of mutations. In this study, we analyzed 22 unreported Japanese patients with Krabbe disease and summarized a total of 51 Japanese patients, including 29 previously reported patients. To elucidate how GALC mutations impair enzymatic activity, multiple disease-causing mutations including common mutations and polymorphisms were investigated for enzymatic activity and precursor processing ability with transient expression system. We also performed 3-D enzyme structure analysis to determine the effect of each new mutation. Five novel mutations were detected including one deletion c.1808delT [p.L603X], one nonsense mutation c.1023C>G [p.Y341X], and three missense mutations c.209T>C [p.L70P], c.1054G>A [p.G352R], and c.1937G>C [p.G646A]. For the total of 51 patients, 59% had late-onset forms of Krabbe disease. Seven common mutations accounted for 58% of mutant alleles of patients with Krabbe disease in Japan. Infantile-onset mutations had almost no enzyme activity, while late-onset mutations had 4%-20% of normal enzyme activity. The processing rate of precursor GALC protein to mature form was slower for infantile-onset mutations. Heat stability of the mutant proteins revealed that p.G270D was more stable compared to the other mutations. The constructed 3D-model showed that the residues for Krabbe mutations were less solvent-accessible and located in the core region of GALC protein. In conclusion, we have demonstrated that the most common phenotype in Japan is the late-onset type, that the enzyme activity for GALC mutants is correlated with mutational severity, and that the most pathogenic factor is due to the processing rate from the precursor to the mature protein.
Keywords: 6-hexadecanoylamino-4-methylumbelliferyl-β-d-galactopyranoside; ASA; BMT; GALC; GalCer; Galactocerebrosidase; Globoid cell leukodystrophy; HM-gal; Krabbe disease; Phenotype; Protein processing; Psychosine; RMSD; TIM; bone marrow transplantation; central triosephosphate isomerase; galactocerebrosidase; galactocerebroside; root-mean-square distance; solvent-accessible surface area.
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