Mutational studies in Korean patients with antithrombin deficiency and cloning of a novel murine acid ceramidase homologue gene

Abstract

Antithrombin is a major physiological inhibitor of serine protease in blood coagulation system. Patients with reduced level of antithrombin in the plasma are predisposed to severe blood clotting disorders. Three cases of patients with antithrombin deficiency were investigated on the molecular level. In case one, a C to T point mutation converting Arg (CGT) 393 to Cys (TGT) was found in exon 6. Due to this point mutation, mutant antithrombin protein formed antithrombin-albumin complex. In case two, a G to C point mutation converting Ala (GCA) 384 to Pro (CCA) was found in exon 6. And in case three, a T to C point mutation substituting Ser (TCA) 365 to Pro (CCA) was found in exon 5. Each point mutation destroys restriction enzyme site, EaeI, PvuII, and DdeI, respectively. A cDNA sequence that encodes for a protein, which is highly homologous to human acid ceramidase (AC; E.C.2.5.1.23) was found from a mouse spleen cDNA library and named as mACH gene. A deficiency of acid ceramidase is known to cause ceramide metabolic disease, Farber lipogranulomatosis. The mACH cDNA (2,455 bp) predicts an open-reading frame coding for a 362 amino acids that was 35% identical and 51% similar to human acid ceramidase. It spans over 20 kb and contains 11 exons separated by 10 introns. Northern blot analysis revealed that mACH is expressed in various tissue, brain, spleen, liver, kidney, lung, intestine, and stomach.