Session: MON 37-82-Pheochromocytoma & Paraganglioma
Poster Board MON-41
Material and methods: Nine patient DNA samples with different known sequence variants in PC/PGL genes (VHL, MEN2, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and MAX) were amplified by long range PCR. Amplicons (~10kb each) were visualized on 1% agarose gels, quantified by Pico Green dsDNA assay, pooled in equimolar ratios and fragmented to 250-500b using sonication. Paired-end libraries were constructed using the TruSeq library assembly kit (Illumina, San Diego, CA). The indexed patient-specific libraries were then multiplexed and run on as either a 2x100bp cycle on a single lane of the Illumina HiSeq 2000 or as a 2x150bp MiSeq run.
Results: Total turn-around time is <5 days, similar to our current SS assays, with all 9 samples run in a single lane, and delivered complete sequences for all 9 genes for all patients at a total analytical cost of <$4000. The samples contained a mix of variations, from single nucleotide variants to multiple nucleotide deletions and a whole exon deletion. Bioinformatic analysis was able to detect all known sequence variants, including a 15 base pair deletion, but was unable to detect a complete deletion of one exon. Results were identical for HiSeq and MiSeq.
Conclusion: By performing an NGS assay for PCC and PGL susceptibility genes, we were able to produce a comprehensive and more complete genetic picture of mutations in nine PC/PGL genes in nine patients, at a cost that is competitive with SS-testing of one or two PC/PGL genes in a single patient. Reporting can be performed for all genes tested, or selectively for any specific combinations of PC/PGL gene(s) that were requested. Additional results can be provided at the press of a button, if required, thus avoiding multiple re-testing of some patients, and preventing additional costs or reporting time delays. However, large deletion detection currently still requires additional testing. NGS is likely to supplant individual SS-based PC/PGL gene testing in all but those patients with known familial mutations.
Nothing to Disclose: DM, SJM, KWC, GV, AA, SKGG
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