Functional splicing assay of DSPP mutations in hereditary dentin defects.

OBJECTIVE: Dentin sialophosphoprotein (DSPP) gene mutations have been identified in isolated hereditary dentin defects; however, the genotype-phenotype correlations are poorly understood. We performed in vitro splicing assays to test the hypothesis that DSPP mutations in splice junctions as well as proposed missense/nonsense mutations experimentally result in aberrant pre-mRNA splicing.
MATERIALS AND METHODS: The genomic fragment of the human DSPP gene was cloned into the pSPL3 splicing vector, and previously reported as well as informative de novo mutations were then introduced by PCR mutagenesis. The COS-7 cells were transfected with each plasmid vector, and total RNA was isolated. RT-PCR result was analyzed, and the band intensity of the product was calibrated using ImageJ.
RESULTS: The predictions by others of exon 3 skipping in specific DSPP mutations have been validated and a cryptic splicing donor site has been identified. However, the degree of mutational effect on pre-mRNA splicing varied considerably depending on the changed nucleotide.
CONCLUSIONS: The predictions of exon 3 skipping in specific DSPP mutations have been validated, and a cryptic splicing donor site has been identified. Our data may provide insight into the contribution of DSPP mutations in the pathogenesis and genotype-phenotype correlations of hereditary dentin defects.