Computational protocol: Impact of connective tissue disease on the surgical outcomes of aortic dissection in patients with cystic medial necrosis

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Protocol publication

[…] Mutation analyses were performed by bidirectional Sanger sequencing of exons and exon-intron boundaries of FBN1, TGFBR1, and TGFBR2 genes first [–]. PCR products were purified and sequenced using BigDye Terminator chemistry v.1.1 on an ABI Prism3130xl or 3730xl (Applied Biosystems). In cases in which mutations of FBN1 gene were not found, mutations were further examined with the multiple ligation probe amplification method on an ABI Prism3130xl (Applied Biosystems). In cases in which these two methods failed to find mutations in patients, SMAD3, ACTA2, and TGFB2 genes were additionally analyzed by bidirectional Sanger sequencing of exons and exon-intron boundaries. For patients without mutations in FBN1, TGFBR1, TGFBR2, SMAD3, ACTA2, or TGFB2 genes, exome sequencing was performed after TruSeq Exome enrichment on HiSeq1000 (Illumina) for searching mutations in MYH11, COL3A1, COLIAI (COL1A1), and COL1A2 genes [–]. Nonsense, missense, or splicing variations in these genes were further analyzed by Sanger sequencing if they were not present in SNP databases, predicted to be damaging by PolyPhen-2, or the SIFT program, or previously reported to be a pathogenic mutation. […]

Pipeline specifications

Software tools PolyPhen, SIFT
Application WES analysis
Organisms Homo sapiens