142 Biochimiea et Biophysiea Aeta, 1182 (t993) 142-146 © 1993 Elsevier Science Publishers B.V. All rights reserved 0925-4439/93/$06.00 BBADIS 61289 Molecular basis of an adult form of Sandhoff disease: Substitution of glutamine for arginine at position 505 of the/3-chain of/3-hexosaminidase results in a labile enzyme Pieter A. Bolhuis, Nico J. Ponne, Hennie Bikker, Frank Baas and J.M.B. Vianney de Jong Academic Medical Center, Department of Neurolo,w, Amsterdam (The Netherlands) (Received 27 August 1992) (Revised manuscript received 15 January 1993) Key words: Hexosaminidase; Sandhoff disease; Genetic compound; Amino acid substitution Sandhoff disease is a lysosomal storage disorder characterized by accumulation of GM2 ganglioside due to mutations in the /3-chain of 13-hexosaminidase. Hexosaminidase activity is negligible in infantilc Sandhoff disease whereas rcsidual 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 genc encoding thc ¢3-chain. Northern analysis of RNA from cultured fibroblasts demonstrated that at least one of the B-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/3-chain, present even in the slime mold Dictyostelium discoideum. The nucleotide transition generated a new restriction site for DdeI, which was present in only one of the alleles of the patient. Reverse transcription of mRNA followed by restriction with DdeI resulted in complete digestion at the mutation site, demonstrating that the second allele 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 exprcssion of a labile form of /3-hexosaminidase. These results show that the patient is a genetic compound, and that the lability of/3-hexosaminidase found in this form of Sandhoff disease is based on a single nucleotidc transition. Introduction Inherited disorders of the lysosomal enzyme /3- hexosaminidase (/3-N-acetyl-D-hexosaminidase, EC 3.2.1.52) were reviewed recently by Neufeld [1] and by Sandhoff et al. [2]. Sandhoff disease is biochemically characterized by decreased activity of the two isoen- zymes, hexosaminidase A, composed of a- and /3-sub- units, and hexosaminidase B, composed of /3-subunits only. Deficiency of the isoenzymes by mutation of the /3-subunit results in accumulation of GM2 ganglioside, in particular in the nervous system. Similar accumula- tions occur by mutation of the a-subunit in Tay-Sachs disease and by mutation of the GM2 activator protein. Correspondence to: P.A. Bolhuis, Academic Medical Center, De- partment of Neurology, 1105 AZ Amsterdam, The Netherlands. Abbreviations: GM2 ganglioside, N-acetylgalactosaminyl-/31---, 4-(N- acetylneuraminyl-o~l ~ 3)-galactosyl-/31 ~ 4-glucosyl-/31 ---,1-ceramide; bp, base pair. The severity of Sandhoff disease is correlated with the decrease in enzyme activity. In the classic (in- fantile) form, the activity of the isoenzymes of /3- hexosaminidase is virtually zero, whereas residual activ- ity has been detected in juvenile and adult forms. Little is known about the ¢3-subunit mutations causing the various forms of Sandhoff disease. Deletion of the 5'-half of the gene was found in one or two alleles of about half the patients with the classic form [3-7] in which also small deletion mutations [8] and point muta- tions [9] have been detected. Intronic and exonic point mutations were found in juvenile forms of the disease [10-121. The first report of adult Sandhoff disease [13] de- scribed two sisters with spinocerebellar degeneration. We demonstrated accumulation of GM2-ganglioside in the central nervous system, labile /3-hexosaminidase in the fibroblasts, and abnormal urinary oligosaccharides in these patients [14]. The molecular basis of this form of Sandhoff disease is the subject of the present study.