Y418C: a novel mutation in exon 9 of the glucocerebrosidase gene of a patient with Gaucher disease creates a new Bgl I site. Received: 22 November 1993 ...
Hum Genet (1994) 94:314-315
9 Springer-Verlag 1994
Renu Tuteja 9 Narendra Tuteja 9 Franco Lilliu Bruno Bembi 9 Renzo Galanello 9 Antonio Cao Francisco E. Baralle
Y418C: a novel mutation in exon 9 of the glucocerebrosidase gene of a patient with Gaucher disease creates a new Bgl I site
Received: 22 November 1993 / Revised 7 February 1994
A b s t r a c t We report a novel mutation in exon 9 of the glucocerebrosidase gene of a patient with Gaucher disease and of Sardinian origin.
Introduction Gaucher disease (GD), a common glycolipid storage disease, is an inherited autosomal recessive disorder characterised by a deficiency of the enzyme glucocerebrosidase (E.C.3.2.1.45) (Beutler 1993). In the framework of our study of the GD mutations present in the Italian population, we wish to report a novel mutation in exon 9 of the glucocerebrosidase gene of a GD patient of Sardinian origin.
Materials and methods The region 5569-6604 of the glucocerebrosidase gene was amplified by the polymerase chain reaction (PCR) from whole blood of the patient as previously described (Tuteja et al. 1993). This patient did not exhibit the L444P mutation (Horowitz et al. 1993) but was found to be heterozygous for the N370S mutation detected by direct sequencing of the PCR product (Fig. 1A).
Results and discussion In order to detect a recently reported polymorphism (Beutler et al. 1992) at position 6144 (G to A), the ampli-
R. Tuteja (l~). N. Tuteja. F. E. Baralle International Centre for Genetic Engineering and Biotechnology-UNIDO, Padriciano 99, Area Science Park, 1-34012 Trieste, Italy F. Lilliu - R. Galanello. A. Cao lstituto di Clinica e Biologia dell'Et~ Evolutiva, Universith degli Studi di Cagliari, Via Jenner, 1-09100 Cagliari, Italy B. Bembi Istituto Burlo Garofolo of Trieste, Via dell'Istria 65/1, 1-34145 Trieste, Italy
fled DNA was digested with BgII.The results showed two bands of 417 and 619bp instead of 575 and 461 bp as reported previously (Tuteja et al. 1993). This observation suggested that there could be another mutation that results in the creation of a new BglI site. The PCR product was cloned in the Smal site of pUC 18 and sequenced. The sequencing data confirmed the presence of the N370S mutation on one allele of the patient. It was further found that the patient was heterozygous for a point mutation changing an A to G (in 5 of 10 clones) at position 1370 (5985 genomic); this causes a replacement of tyrosine 418 to cysteine (Y418C) (Fig. I B). A recognition site for Bgll is created by this mutation, making it possible to test quickly for the mutation using genomic DNA amplification and restriction enzyme cleavage. It was found that the mutation occurred in only one of the 24 alleles from our GD patients and in none of the 50 alleles from tested normal controls. The region 34654101 of the glucocerebrosidase gene was also amplified (Tuteja et al. 1993) to study the common PvuII polymorphism and its association with the new mutation. The patient was found to be homozygous for the Pv 11- genotype, showing that the Y418C mutation in this case is on the P v l l - h a p l o t y p e , a s is the previously described N370S mutation (Beutler 1993). Both mutations N370S and Y418C are in exon 9 of the gene (Fig. 1C). Mutation analysis of the genomic DNA from the parents of the patient shows that the patient has inherited the N370S mutation from the mother and the Y418C mutation from the father. Glucocerebrosidase expressed by the N370S mutant allele has a decreased activity (Grace et al. 1990). The analysis described above strongly suggests that the tyrosine to cysteine change in the protein at position 418 is the disease-causing mutation. Indeed, the active-site functional domains of glucocerebrosidase are carboxy-terminal, and the region between amino-acids 315 and 440 has 97% identity between mouse and human. Tyrosine 418 and 100% of the cysteines in particular are conserved (O'Neill et al. 1989). The Y418C mutation adds one more cysteine to the protein in this critical region; this change probably alters the folding of the protein and adversely af-
315 Fig. 1 A Direct genomic DNA sequence of exon 9 around the mutation site. The heterozygous A--4G lesion and the predicted amino acid substitution in one letter code at position 370 is shown. B Sequence from one clone of each allele from the patient. The mutated base is underlined and the predicted amino acid substitution at position 418 is shown. C Partial map of the functional glucocerebrosidase gene covering exons 9 to 11. The mutations (arrows) indicate the positions of the amino acid changes (asparagine 370 to serine, and tyrosine 418 to cysteine). In both mutations, the nucleotide change is A---~G
fects its stability and/or enzymatic activity. It is also possible that the Y418C mutant enzyme is poorly stimulated by a natural activator saposin C, as has been shown for another mutated enzyme, R463C (Ohashi et al. 1991).
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