A Next Generation Sequencing Nine-Gene Test Panel for Pheochromocytoma and Paraganglioma

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 37-82-Pheochromocytoma & Paraganglioma
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-41
Dragana Milosevic*, Stephen J Murphy, Kendall W Cradic, George Vasmatzis, Alicia Algeciras-Schimnich and Stefan Karl Gunther Grebe
Mayo Clinic and Foundation, Rochester, MN
Introduction: Hereditary pheochromocytoma and paraganglioma (PC/PGL) syndromes are diagnosed by physical examination, family history, imaging studies, biochemical testing, and genetic testing. Sanger-Sequencing (SS) is used for an increasing number of known susceptibility genes. Since testing all these genes is prohibitively expensive and time consuming, a range of different clinical/biochemical decision making algorithms are used to prioritize the testing order. Despite this, several genes frequently end up being tested, resulting in a labor/time intensive and expensive work-up. We therefore created a Next Generation Sequencing (NGS) panel that allows simultaneous NGS of several bar-coded/indexed patient samples for mutations in most PCC/PGL genes. The method offers time and cost savings and potentially allows for improved mutation detection sensitivity in cases of mosaicism, or when examining tumor tissues.

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

*Please take note of The Endocrine Society's News Embargo Policy at http://www.endo-society.org/endo2013/media.cfm