Molecular basis of an adult form of Sandhoff disease: Substitution of glutamine for arginine at position 505 of the β-chain of β-hexosaminidase results in a labile enzyme

Abstract

Sandhoff disease is a lysosomal storage disorder characterized by accumulation of G M2 ganglioside due to mutations in the β-chain of β-hexosaminidase. Hexosaminidase activity is negligible in infantile Sandhoff disease whereas residual activity is present in juvenile and adult forms. Here we report the molecular basis of the first described adult form of Sandhoff disease. Southern analysis of chromosomal DNA indicated the absence of chromosomal deletions in the gene encoding the β-chain. Northern analysis of RNA from cultured fibroblasts demonstrated that at least one of the β-chain alleles was transcribed into normal-length mRNA. Sequence analysis of the entire cDNA prepared from poly-adenylated RNA showed that only one point mutation was present, consisting of a G→A transition at nucleotide position 1514. This mutation changes the electric charge at amino acid position 505 by substitution of glutamine for arginine in a highly conserved part of the β-chain, present even in the slime mold Dictyostelium discoideum. The nucleotide transition generated a new restriction site for Dde I, which was present in only one of the alleles of the patient. Reverse transcription of mRNA followed by restriction with Dde I resulted in complete digestion at the mutation site, demonstrating that the secone allel was of an mRNA-negative type. Transfection of COS cells with a cDNA construct containing the mutation but otherwise the normal sequence resulted in the expression of a labile form of β-hexosaminidase. These results show that the patient is a genetic compound, and that the lability of β-hexosaminidase found in this form of Sandhoff disease is based on a single nucleotide transition.