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Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis |
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| Authors: | Chia-Cheng Hung Shin-Yu Lin Chien-Nan Lee Chiou-Ya Lin Hsin-Hui Chiu Shuan-Pei Lin Hong-Nerng Ho Yi-Ning Su |
| Affiliation: | a Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan b Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan c Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan d Institute of Molecular Medicine, National Taiwan University College of Medicine, Taipei, Taiwan e Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan f Department of Integrated Diagnostics and Therapeutics, National Taiwan University Hospital, Taipei, Taiwan g Division of Genetics and Metabolism, Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan h Department of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan i Graduate Institute of Clinical Genomics, National Taiwan University College of Medicine, Taipei, Taiwan |
| Abstract: | Marfan syndrome has been associated with approximately 562 mutations in the fibrillin-1 (FBN1) gene. Mutation scanning of the FBN1 gene with DNA direct sequencing is time-consuming and expensive because of its large size. This study analyzed the diagnostic value of high-resolution melting analysis as an alternative method for scanning of the FBN1 gene. A total of 75 polymerase chain reaction (PCR) amplicons (179-301 bp, average 256 bp) that covered the complete coding regions and splicing sites were evaluated on the 96-well LightCycler system. Melting curves were analyzed as fluorescence derivative plots (−dF/dT vs. temperature). To determine the sensitivity of this method, a total of 82 samples from patients with Marfan syndrome and 50 unaffected individuals were analyzed. All mutations reported in this study had been confirmed previously by direct sequencing analysis. Melting analysis identified 48 heterozygous variants. The variant c.3093 G>T (exon 25) was incorrectly identified by melting curve analysis. The sensitivity of the technique in this sample was 98.78% (81/82). This study demonstrated that high-resolution melting analysis is a reliable gene scanning method with greater speed than DNA sequencing. Our results support the use of this technology as an alternative method for the diagnosis of Marfan syndrome as well as its suitability for high-throughput mutation scanning of other large genes. |
| Keywords: | Marfan syndrome FBN1 gene High-resolution melting (HRM) |
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