Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis.
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 (179-301bp, average 256bp) 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.
Hung
Chia-Cheng
CC
Department of Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan.
Lin
Shin-Yu
SY
Lee
Chien-Nan
CN
Cheng
Hui-Yu
HY
Lin
Chiou-Ya
CY
Chang
Chien-Hui
CH
Chiu
Hsin-Hui
HH
Yu
Chih-Chieh
CC
Lin
Shuan-Pei
SP
Cheng
Wen-Fang
WF
Ho
Hong-Nerng
HN
Niu
Dau-Ming
DM
Su
Yi-Ning
YN