The human acid ceramidase gene (ASAH): Structure, chromosomal location, mutation analysis, and expression

Acid ceramidase (AC) is the lysosomal enzyme that degrades ceramide into sp hingosine and fatty acid. A deficiency in human AC activity leads to the ly sosomal storage disorder, Farber disease (FD). The human AC gene (HGMW-appr oved symbol ASAH) was cloned and characterized, revealing an organization s imilar to that of the murine AC gene. The human gene spans about 30 kb in l ength and contains 14 exons ranging in size from 46 to 1201 bp. The exon/in tron junctions were determined and found to follow the GT-AG; rule. The put ative promoter region had a GC content over 60%, lacked a TATA box, and con tained several sequences matching transcription factor binding sites, inclu ding nine SP-1 sites, one AP-1 site, and three CACC boxes. The promoter act ivity of a 475-bp fragment from within this region was demonstrated by chlo ramphenicol acyltransferase assays. Northern blotting revealed variable exp ression of the human AC RNA; i.e., expression of the major 2.4-kb transcrip t was high in heart and kidney, followed by lung and placenta, but low in p ancreas, liver, brain, and skeletal muscle. Two minor AC transcripts of 1.7 and 1.2 kb also were detected in heart and skeletal muscle. The human AC g ene was mapped to the chromosomal region 8p21.3-p22 by in situ hybridizatio n and FISH analyses, syntenic with the mouse chromosomal location. Finally, three new missense mutations, E138V, R254G, and P362R, mere identified in the human AC gene from FD patients. Mutant AC cDNAs containing these point mutations were constructed and examined using the FLAG-tagged expression sy stem. Although the levels of protein expression for these mutant ACs were a bout equivalent to that of the controls, their enzymatic activity was marke dly reduced, confirming their authenticity. (C) 1999 Academic Press.