Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes
- St. Louis Univ. School of Medicine, MO (United States)
PCR of cDNA produced from patient fibroblasts allowed the authors to determine the paternal mutation in the first patient reported with [beta]-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G[r arrow]T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reduced stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, the authors found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed them to amplify and characterize genomic sequences for the true [beta]-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C[r arrow]T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression. 39 refs., 5 figs., 1 tab.
- OSTI ID:
- 6574557
- Journal Information:
- American Journal of Human Genetics; (United States), Vol. 52:3; ISSN 0002-9297
- Country of Publication:
- United States
- Language:
- English
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GENE MUTATIONS
DETECTION
GLUCURONIDASE
GENES
MUCOPOLYSACCHARIDES
METABOLIC DISEASES
DNA SEQUENCING
HUMAN CHROMOSOME 22
HUMAN CHROMOSOME 5
HUMAN CHROMOSOMES
HUMAN Y CHROMOSOME
AMINES
CARBOHYDRATES
CHROMOSOMES
DISEASES
ENZYMES
GLYCOSYL HYDROLASES
HETEROCHROMOSOMES
HYDROLASES
MUTATIONS
O-GLYCOSYL HYDROLASES
ORGANIC COMPOUNDS
POLYSACCHARIDES
PROTEINS
SACCHARIDES
STRUCTURAL CHEMICAL ANALYSIS
Y CHROMOSOME
550400* - Genetics