title: Functional splicing assay of DSPP mutations in hereditary dentin defects.
abstract: 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.
  
  
  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.
authors: Department of Pediatric Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea.
authors: K-E
authors: KE
authors: Lee
authors: S-K
authors: SK
authors: Lee
authors: S-E
authors: SE
authors: Jung
authors: Zh
authors: Zh
authors: Lee
authors: J-W
authors: JW
authors: Kim
mesh_terms: D000225:Adenine; D018389:Codon, Nonsense / Q000235:genetics; D017124:Conserved Sequence / Q000235:genetics; D003596:Cytosine; D004252:DNA Mutational Analysis; D003805:Dentin Dysplasia / Q000235:genetics*; D003811:Dentinogenesis Imperfecta / Q000235:genetics*; D005091:Exons / Q000235:genetics; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005838:Genotype; D006147:Guanine; D006801:Humans; D007532:Isoleucine / Q000235:genetics; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D020125:Mutation, Missense / Q000235:genetics; D010641:Phenotype; D010750:Phosphoproteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011392:Proline / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012795:Sialoglycoproteins / Q000235:genetics*; D013941:Thymine; D014633:Valine / Q000235:genetics; D000099311:Dentin Sialophosphoprotein
chemical_list: D000225:Adenine; D018389:Codon, Nonsense; D003596:Cytosine; D016326:Extracellular Matrix Proteins; D006147:Guanine; D007532:Isoleucine; D010750:Phosphoproteins; D011392:Proline; D022821:RNA Splice Sites; D012795:Sialoglycoproteins; D013941:Thymine; D014633:Valine; D000099311:Dentin Sialophosphoprotein