Approximately 90% of patients with osteogenesis imperfecta (OI) exhibit dominant COL1A1 or COL1A2 mutations; however, molecular analysis is difficult because these genes span 51 and 52 exons, respectively. We devised a PCR-denaturing high-performance liquid chromatography (DHPLC) procedure to analyze the COL1A1 or COL1A2 coding regions and validated it using 130 DNA samples from individuals without OI, 25 DNA samples from two cells to investigate the procedure's potential for preimplantation diagnosis, and DNA samples from 10 patients with OI. Three novel intronic variants in vitro were expressed using a minigene assay to assess their effects on splicing. The procedure is rapid, inexpensive, and reproducible. Analysis of samples from individuals without OI revealed six novel and some known polymorphisms useful for linkage diagnosis because of high heterozygosity. Analysis of two-cell samples confirmed the known genotype in 24 of 25 experiments; DNA failed to amplify in only one case. No incidence of allele dropout was recorded. DHPLC revealed six novel mutations, three of which were intronic, in all patients with OI, and these results were confirmed by means of COL1A1 and COL1A2 direct sequencing. Expression of intronic mutations demonstrated that variant 804 + 2_804 + 3delTG in intron 11 disrupts normal splicing, thereby leading to formation of two alternative products. Variants c.3046-4_3046-5dupCT (COL1A1) and c.891 + 77A>T (COL1A2) did not affect splicing. The described DHPLC protocol combined with the minigene assay may contribute to molecular diagnosis in OI. Moreover, this protocol will aid in counseling about prenatal and preimplantation diagnosis.

Approximately 90% of patients with osteogenesis imperfecta (00 exhibit dominant COL1A1 or COL1A2 mutations; however, molecular analysis is difficult because these genes span 51 and 52 exons, respectively. We devised a PCR-denaturing high-performance liquid chromatography (DHPLC) procedure to analyze the COL1A1 or COL1A2 coding regions and validated it using 130 DNA samples from individuals without OI, 25 DNA samples from two cells to investigate the procedure's potential for preimplantation diagnosis, and DNA samples from 10 patients with OI. Three novel intronic variants in vitro were expressed using a minigene assay to assess their effects on splicing. The procedure is rapid, inexpensive, and reproducible. Analysis of samples from individuals without OI revealed six novel and some known polymorphisms useful for linkage diagnosis because of high heterozygosity. Analysis of two-cell samples confirmed the known genotype in 24 of 25 experiments; DNA failed to amplify in only one case. No incidence of allele dropout was recorded. DHPLC revealed six novel mutations, three of which were intronic, in all patients with OI, and these results were confirmed by means of COL1A1 and COL1A2 direct sequencing. Expression of intronic mutations demonstrated that variant 804 + 2_804 + 3delTG in intron 11 disrupts normal splicing, thereby leading to formation of two alternative products. Variants c.3046-4_3046-5dupCT (COL1A1) and c.891 + 77A>T (COL1A2) did not affect splicing. The described DHPLC protocol combined with the minigene assay may contribute to molecular diagnosis in OI. Moreover, this protocol will aid in counseling about prenatal and preimplantation diagnosis. (J Mol Diagn 2011, 13:648-654. DOI: 10.1016/j.jmoldx.2011.06.006)

A novel DHPLC-based procedure for the analysis of COL1A1 and COL1A2 mutations in osteogenesis imperfecta / Fuccio, Antonella; Iorio, Mariangela; Amato, Felice; Elce, Ausilia; Ingino, Rosaria; Filocamo, Mirella; Castaldo, Giuseppe; Salvatore, Francesco; Tomaiuolo, Rossella. - In: THE JOURNAL OF MOLECULAR DIAGNOSTICS. - ISSN 1525-1578. - 13:6(2011), pp. 648-656. [10.1016/j.jmoldx.2011.06.006]

A novel DHPLC-based procedure for the analysis of COL1A1 and COL1A2 mutations in osteogenesis imperfecta

Tomaiuolo, Rossella
Ultimo
2011-01-01

Abstract

Approximately 90% of patients with osteogenesis imperfecta (00 exhibit dominant COL1A1 or COL1A2 mutations; however, molecular analysis is difficult because these genes span 51 and 52 exons, respectively. We devised a PCR-denaturing high-performance liquid chromatography (DHPLC) procedure to analyze the COL1A1 or COL1A2 coding regions and validated it using 130 DNA samples from individuals without OI, 25 DNA samples from two cells to investigate the procedure's potential for preimplantation diagnosis, and DNA samples from 10 patients with OI. Three novel intronic variants in vitro were expressed using a minigene assay to assess their effects on splicing. The procedure is rapid, inexpensive, and reproducible. Analysis of samples from individuals without OI revealed six novel and some known polymorphisms useful for linkage diagnosis because of high heterozygosity. Analysis of two-cell samples confirmed the known genotype in 24 of 25 experiments; DNA failed to amplify in only one case. No incidence of allele dropout was recorded. DHPLC revealed six novel mutations, three of which were intronic, in all patients with OI, and these results were confirmed by means of COL1A1 and COL1A2 direct sequencing. Expression of intronic mutations demonstrated that variant 804 + 2_804 + 3delTG in intron 11 disrupts normal splicing, thereby leading to formation of two alternative products. Variants c.3046-4_3046-5dupCT (COL1A1) and c.891 + 77A>T (COL1A2) did not affect splicing. The described DHPLC protocol combined with the minigene assay may contribute to molecular diagnosis in OI. Moreover, this protocol will aid in counseling about prenatal and preimplantation diagnosis. (J Mol Diagn 2011, 13:648-654. DOI: 10.1016/j.jmoldx.2011.06.006)
2011
Approximately 90% of patients with osteogenesis imperfecta (OI) exhibit dominant COL1A1 or COL1A2 mutations; however, molecular analysis is difficult because these genes span 51 and 52 exons, respectively. We devised a PCR-denaturing high-performance liquid chromatography (DHPLC) procedure to analyze the COL1A1 or COL1A2 coding regions and validated it using 130 DNA samples from individuals without OI, 25 DNA samples from two cells to investigate the procedure's potential for preimplantation diagnosis, and DNA samples from 10 patients with OI. Three novel intronic variants in vitro were expressed using a minigene assay to assess their effects on splicing. The procedure is rapid, inexpensive, and reproducible. Analysis of samples from individuals without OI revealed six novel and some known polymorphisms useful for linkage diagnosis because of high heterozygosity. Analysis of two-cell samples confirmed the known genotype in 24 of 25 experiments; DNA failed to amplify in only one case. No incidence of allele dropout was recorded. DHPLC revealed six novel mutations, three of which were intronic, in all patients with OI, and these results were confirmed by means of COL1A1 and COL1A2 direct sequencing. Expression of intronic mutations demonstrated that variant 804 + 2_804 + 3delTG in intron 11 disrupts normal splicing, thereby leading to formation of two alternative products. Variants c.3046-4_3046-5dupCT (COL1A1) and c.891 + 77A>T (COL1A2) did not affect splicing. The described DHPLC protocol combined with the minigene assay may contribute to molecular diagnosis in OI. Moreover, this protocol will aid in counseling about prenatal and preimplantation diagnosis.
Base Sequence
Chromatography, High Pressure Liquid
Collagen Type I
Collagen Type I, alpha 1 Chain
DNA Mutational Analysis
Gene Frequency
Genotype
Humans
Osteogenesis Imperfecta
Polymerase Chain Reaction
Polymorphism, Single Nucleotide
Preimplantation Diagnosis
RNA Splicing
Sequence Analysis, DNA
Molecular Diagnostic Techniques
Mutation
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11768/133937
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