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[Detection of pathogenic mutations in Marfan syndrome by targeted next-generation semiconductor sequencing]
Chaoxia Lu1, Wei Wu, Jifang Xiao
1McKusick-Zhang Center for Genetic Medicine, Institute of Basic Medical Sciences, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005, P.R. China.
Objective:
To detect pathogenic mutations in Marfan syndrome (MFS) using an Ion Torrent Personal Genome Machine (PGM) and to validate the result of targeted next-generation semiconductor sequencing for the diagnosis of genetic disorders.
Methods:
Peripheral blood samples were collected from three MFS patients and a normal control with informed consent. Genomic DNA was isolated by standard method and then subjected to targeted sequencing using an Ion Ampliseq(TM) Inherited Disease Panel. Three multiplex PCR reactions were carried out to amplify the coding exons of 328 genes including FBN1, TGFBR1 and TGFBR2. DNA fragments from different samples were ligated with barcoded sequencing adaptors. Template preparation and emulsion PCR, and Ion Sphere Particles enrichment were carried out using an Ion One Touch system. The ion sphere particles were sequenced on a 318 chip using the PGM platform. Data from the PGM runs were processed using an Ion Torrent Suite 3.2 software to generate sequence reads. After sequence alignment and extraction of SNPs and indels, all the variants were filtered against dbSNP137. DNA sequences were visualized with an Integrated Genomics Viewer. The most likely disease-causing variants were analyzed by Sanger sequencing.
Results:
The PGM sequencing has yielded an output of 855.80 Mb, with a > 100 × median sequencing depth and a coverage of > 98% for the targeted regions in all the four samples. After data analysis and database filtering, one known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X and p.L871FfsX23) in the FBN1 gene were identified in the three MFS patients. All mutations were verified by conventional Sanger sequencing.
Conclusion:
Pathogenic FBN1 mutations have been identified in all patients with MFS, indicating that the targeted next-generation sequencing on the PGM sequencers can be applied for accurate and high-throughput testing of genetic disorders.
Insights
Pathogenic FBN1 gene mutations were detected in Marfan syndrome (MFS) patients using Ion Torrent PGM sequencing. This targeted next-generation sequencing method proves effective for high-throughput genetic disorder diagnosis.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Marfan syndrome (MFS) is a genetic disorder affecting connective tissue.
- Accurate genetic diagnosis is crucial for MFS management.
- Next-generation sequencing (NGS) offers a powerful tool for genetic mutation detection.
Purpose of the Study:
- To identify pathogenic mutations in the FBN1 gene in Marfan syndrome patients.
- To evaluate the efficacy of Ion Torrent Personal Genome Machine (PGM) for targeted sequencing.
- To validate targeted NGS for diagnosing genetic disorders.
Main Methods:
- Genomic DNA was isolated from peripheral blood of MFS patients and controls.
- Targeted sequencing was performed using an Ion Ampliseq Inherited Disease Panel covering 328 genes.
- Data analysis involved sequence alignment, variant filtering, and Sanger sequencing for validation.
Main Results:
- PGM sequencing achieved high depth (>100x) and coverage (>98%) for targeted regions.
- One known missense mutation (p.E1811K) and two novel premature termination mutations (p.E2264X, p.L871FfsX23) in FBN1 were identified.
- All identified mutations were confirmed by Sanger sequencing.
Conclusions:
- Pathogenic FBN1 mutations were found in all MFS patients studied.
- Targeted NGS on PGM sequencers is suitable for accurate, high-throughput genetic disorder testing.
- This approach facilitates precise diagnosis and potential therapeutic strategies for MFS.
