title: Identification of novel splice site mutation IVS9 + 1(G > A) and novel complex allele G355R/R359X in Type 1 Gaucher patients heterozygous for mutation N370S.
abstract: Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity. More than 350 mutations that cause Gaucher disease have been described to date. Novel mutations can potentially provide insight into the glucocerebrosidase structure-function relationship and biochemical basis of the disease. Here, we report the identification of two novel mutations in two unrelated patients with type I (non-neuronopathic) Gaucher disease: 1) a splice site mutation IVS9 + 1G > A; and (2) a complex allele (cis) G355R/R359X. Both patients have a common N370S mutation in the other allele. The splice site mutation results from an intronic base substitution (G to A, c.1328 + 1, g.5005) at the donor splice site of exon and intron 9. The complex allele results from two point mutations in exon 8 of glucocerebrosidase (G to C at c.1180, g.4396, and T to C at c. 1192, g.4408) substituting glycine by arginine (G355R) and arginine by a premature termination (R359X), respectively. In order to demonstrate that G355R/R359X are in cis arrangement, PCR-amplified glucocerebrosidase exon 8 genomic DNA from the patient was cloned into the vector pJET1.2 in Escherichia coli TOP10® strain. Out of the 15 clones that were sequence analyzed, 10 contained the normal allele sequence and 5 contained the complex allele G355R/R359X sequence showing both mutations in cis arrangement. Restriction fragment length polymorphism analysis using Hph1 restriction endonuclease digest was established for the IVS9 + 1G > A mutation for confirmation and efficient identification of this mutation in future patients. Past literature suggests that mutations affecting splicing patterns of the glucocerebrosidase transcript as well as mutations in Gaucher complex alleles are detrimental to enzyme activity. However, compound heterozygosity with N370S, a mild mutation, will lead to a mild phenotype. The cases reported here support these past findings.
authors.affiliation: Department of Biology, Centre for Biomedical Research, University of Victoria, British Columbia, Canada.
authors.forename: Kourtnee
authors.initials: K
authors.lastname: Hoitsema
authors.affiliation: Mark Freedman and Judy Jacobs Program for Gaucher Disease, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Dominick
authors.initials: D
authors.lastname: Amato
authors.affiliation: Department of Medical Genetics, University of Calgary, Alberta Children's Hospital, Calgary, Alberta, Canada; Department of Pediatrics, University of Calgary, Alberta Children's Hospital, Calgary, Alberta, Canada.
authors.forename: Aneal
authors.initials: A
authors.lastname: Khan
authors.affiliation: Department of Medicine, Division of Endocrinology, University of British Columbia, Canada.
authors.forename: Sandra
authors.initials: S
authors.lastname: Sirrs
authors.affiliation: Department of Biology, Centre for Biomedical Research, University of Victoria, British Columbia, Canada.
authors.forename: Francis Y M
authors.initials: FY
authors.lastname: Choy
keywords: Complex alleles; EC, enzyme commission number; GBA novel mutations; GBA, glucocerebrosidase enzyme protein; GBA, glucocerebrosidase gene; GD, Gaucher disease; Gaucher disease; Glucocerebrosidase; Hb, hemoglobin; MRI, magnetic resonance imaging; OMIM, Online Mendelian Inheritance in Man entry number; PCR, polymerase chain reaction; RFLP analysis; RFLP, restriction fragment length polymorphism; RT-PCR, reverse transcription-PCR; Sequence analysis; bp, base pairs; pJET cloning