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Functional splicing assay of DSPP mutations in hereditary dentin defects.
Oral Dis. 2011 Oct; 17(7):690-5.OD

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.

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.

Authors+Show Affiliations

Department of Pediatric Dentistry and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

21736673

Citation

Lee, K-E, et al. "Functional Splicing Assay of DSPP Mutations in Hereditary Dentin Defects." Oral Diseases, vol. 17, no. 7, 2011, pp. 690-5.
Lee KE, Lee SK, Jung SE, et al. Functional splicing assay of DSPP mutations in hereditary dentin defects. Oral Dis. 2011;17(7):690-5.
Lee, K. E., Lee, S. K., Jung, S. E., Lee, Z. h., & Kim, J. W. (2011). Functional splicing assay of DSPP mutations in hereditary dentin defects. Oral Diseases, 17(7), 690-5. https://doi.org/10.1111/j.1601-0825.2011.01825.x
Lee KE, et al. Functional Splicing Assay of DSPP Mutations in Hereditary Dentin Defects. Oral Dis. 2011;17(7):690-5. PubMed PMID: 21736673.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Functional splicing assay of DSPP mutations in hereditary dentin defects. AU - Lee,K-E, AU - Lee,S-K, AU - Jung,S-E, AU - Lee,Zh, AU - Kim,J-W, Y1 - 2011/07/08/ PY - 2011/7/9/entrez PY - 2011/7/9/pubmed PY - 2012/1/27/medline SP - 690 EP - 5 JF - Oral diseases JO - Oral Dis VL - 17 IS - 7 N2 - 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. SN - 1601-0825 UR - https://www.unboundmedicine.com/medline/citation/21736673/Functional_splicing_assay_of_DSPP_mutations_in_hereditary_dentin_defects_ L2 - https://doi.org/10.1111/j.1601-0825.2011.01825.x DB - PRIME DP - Unbound Medicine ER -