A new missense mutation in glucocerebrosidase exon 9 of a non-Jewish Caucasian type 1 Gaucher disease patient.

Gaucher disease is the most prevalent inherited sphingolipidosis (1). It results from deficient glucocerebrosidase activity and is transmitted as an autosomal recessive trait (2). Three clinical forms of Gaucher disease have been described: Type 1, nonneuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic (1). The gene frequency of Gaucher disease in the Jewish population was elevated and estimated to be between 0.035 to 0.040 (3). Four 'public' mutations were reported to account for about 96% of the Gaucher disease-producing alleles in Jewish patients (3). These are an A —G transition at cDNA nt 1226 that results in Asn-Ser (4), a G insertion at cDNA nt 84 (5), a T C transition at cDNA nt 1448 that results in ^Leu—"^Pro (6), and a splicing (g—a) mutation in intron 2 at genomic nt 1067 (5,7). Among non-Jewish patients, it was reported (8) that mutations 1448 and 1226 account for more than 50% of the total mutated alleles. However, more than 25% of the total mutations among non-Jewish patients remain unidentified (3,8,9). In this report, we describe a new missense mutation in a non-Jewish Caucasian type 1 Gaucher patient. A relatively simple procedure which utilizes the polymerase chain reaction (PCR) method and restriction fragment length polymorphism (RFLP) analysis for its detection is also described. The patient is of French —Finnish and French—Ukrainian descent. She was investigated at age 6 1/2 years for unexplained splenomegaly. There was no history of bone pain or neurological problems. The diagnosis of Gaucher disease was raised. Bone marrow aspirate and fibroblast culture for glucocerebrosidase activity assay confirmed the clinical diagnosis of Gaucher disease. She was readmitted at age 13 1/2 years because of hypersplenism and left flank pain. The spleen was large and hard, filling the whole left abdomen. She underwent a splenectomy which reversed her hematological complications. Histology on her spleen confirmed the diagnosis of Gaucher disease. At the present age of 21 years, she was asymptomatic and there was no clinical evidence of hepatic or neurological involvement. Fibroblasts were cultured and harvested for /?-glucosidase activity assays as previously described (10). Genomic DNA was isolated from harvested fibroblasts using the TurboGenTM Genomic DNA Isolation Kit [Invitrogen Corporation, San Diego, CA]. The genomic DNA samples were amplified by the PCR method with Taq polymerase (Bethesda Research Laboratory, Gaithersburg, MD), using a method (11) modified from Saiki el al. (12). In order to selectively amplify the glucocerebrosidase structural gene and not the pseudogene which shares more than 96% sequence similarity with the structural gene (13), primers with nucleotide sequences deleted in the pseudogene were synthesized and used for the PCR amplification [(14), Table 1]. Primers A and B flank the anterior part of exon 9 where mutation 1226 is located. The posterior part of exon 9 (where mutation 1366 is located), exon 10 and exon 11 are flanked by primers C and D. Poly-A mRNA was isolated from cultured fibroblasts using the Micro-FastTrackTM mRNA Isolation Kit (Invitrogen Corporation). The cDNA of the glucocerebrosidase gene was synthesized by reversed transcription of fibroblast poly-A mRNAs using the cDNA CycleTM Kit (Invitrogen Corporation), and amplified by the PCR method as previously described (14). The nucleotide sequences of the primers for the first strand cDNA synthesis (primer D) and PCR amplification (primers D and E) are shown in Table 1. These primers are designed to cover the full length cDNA of glucocerebrosidase (14). Sequence analysis was performed using the dideoxynucleotide chain termination method by Sanger et al. (15) and the fmolTMDNA Sequencing System (Promega Corporation, Madison, WI). The PCR mismatch method and Xhol RFLP analysis (9) were used to detect mutation 1226. For mutation 1366, genomic DNA amplified by the PCR method using primers C and F (Table 1) was subjected to Ncol RFLP analysis.