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Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes
J M Shipley1, M Klinkenberg, B M Wu
1Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104.
Abstract:
PCR of cDNA produced from patient fibroblasts allowed us to determine the paternal mutation in the first patient reported with beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G-->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, we found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed us 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-->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, suggesting that high concentrations of mutant monomers can drive the folding and tetramerization of mutant enzyme to produce an active and stable enzyme.
Insights
Researchers identified two mutations in beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). One mutation resulted in significant enzyme activity, suggesting it may be treatable with gene therapy.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- Mucopolysaccharidosis type VII (MPS VII) is a rare genetic disorder caused by beta-glucuronidase deficiency.
- Identifying specific mutations is crucial for understanding disease mechanisms and developing targeted therapies.
Purpose of the Study:
- To identify and characterize the paternal and maternal mutations in a patient with MPS VII.
- To investigate the functional consequences of identified mutations on beta-glucuronidase enzyme activity.
Main Methods:
- Polymerase Chain Reaction (PCR) amplification and sequencing of cDNA and genomic DNA from patient fibroblasts.
- Restriction fragment digestion analysis (MboII).
- Expression studies in COS-7 and murine MPS VII cells.
Main Results:
- Identified a paternal missense mutation (W627C) in exon 12 and a maternal nonsense mutation (R356STOP) in exon 7.
- The paternal mutation (W627C) resulted in significant residual enzyme activity (65% of control in COS-7 cells, 13% in MPS VII cells), dependent on overexpression.
- The maternal mutation (R356STOP) led to a complete loss of enzyme activity.
Conclusions:
- The identified mutations provide insight into the genetic basis of MPS VII in this patient.
- The residual activity of the paternal mutation suggests potential therapeutic strategies targeting this specific mutation, possibly through gene augmentation or overexpression approaches.