Functional splicing assay of DSPP mutations in hereditary dentin defects.

[dentinogenesis imperfecta]

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-m RNA splicing .T he genomic fragment of the human DSPP gene was cloned into the pSPL 3 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 .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-m RNA splicing varied considerably depending on the changed nucleotide .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 .

Diseases
Validation

Diseases presenting "proposed missense/nonsense mutations" symptom

dentin dysplasia

dentinogenesis imperfecta

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