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J Mol Diagn. 2017 Jul;19(4):575-588. doi: 10.1016/j.jmoldx.2017.04.009. Epub 2017 May 25.

PheoSeq: A Targeted Next-Generation Sequencing Assay for Pheochromocytoma and Paraganglioma Diagnostics.

Author information

1
Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre, Madrid, Spain.
2
Translational Bioinformatics Unit, Spanish National Cancer Research Centre, Madrid, Spain.
3
Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universitat Dresden, Dresden, Germany.
4
Department of Experimental and Clinical Biomedical Sciences Mario Serio, University of Florence and Istituto Toscano Tumori, Florence, Italy.
5
Department of Internal Medicine IV Campus Innenstadt, University-Hospital, Ludwig-Maximilians-University of Munich, Munich, Germany.
6
Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, Würzburg, Germany.
7
Department of Endocrinology and Nutrition Service, University Hospital 12 de Octubre, Madrid, Spain.
8
Department of Endocrinology, University Hospital La Paz, Madrid, Spain.
9
Department of Endocrinology, Albacete University Hospital Complex, Albacete, Spain.
10
Department of Endocrinology, University Hospital Puerta de Hierro, Madrid, Spain.
11
Department of Endocrinology, Virgen de la Salud Hospital-Toledo Hospital Complex, Toledo, Spain.
12
Molecular Diagnostics Units of the Hereditary Cancer Program at the Catalan Institute of Oncology, Barcelona, Spain.
13
Department of Endocrinology, University of Navarra Clinic, Navarra, Spain.
14
Department of Pediatrics and Clinical Genetics Unit, University of Navarra Clinic, Navarra, Spain.
15
Department of Endocrinology, Reina Sofia University Hospital, Murcia, Spain.
16
High Risk and Cancer Prevention Group, Medical Oncology Department, Vall d'Hebron University Hospital and Vall d'Hebron Institute of Oncology, Barcelona, Spain.
17
Department of Endocrinology, San Carlos (Clínico) Hospital, Madrid, Spain.
18
Department of Molecular Oncology, Central University Hospital of Asturias and University Institute of Oncology of Asturias, University of Oviedo, Oviedo, Spain.
19
Department of Endocrinology and Nutrition, University Hospital Arnau de Vilanova, IRBLLEIDA, Lleida, Spain; Department of Pathology, Hospital Universitari de Bellvitge, IDIBELL, Barcelona, Spain.
20
Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre, Madrid, Spain; Biomedical Research Networking Center on Rare Diseases (CIBERER), Madrid, Spain.
21
1st Department of Medicine, University Medical Center Schleswig-Holstein, Campus Lübeck, Lübeck, Germany.
22
Department of Endocrinology, Department of Medical and Surgical Sciences University of Padova, Padova, Italy.
23
Section on Medical Neuroendocrinology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH, Bethesda, Maryland.
24
Department of Pathology, Erasmus University Medical Center, Rotterdam, the Netherlands.
25
Department of Pathology, Erasmus University Medical Center, Rotterdam, the Netherlands; Department of Pathology, Reinier de Graaf Hospital, Delft, the Netherlands.
26
Department of Endocrinology, Centre Hospitalier Universitaire de Liège, Liège, Belgium.
27
Institute of Clinical Chemistry and Laboratory Medicine, University Hospital Carl Gustav Carus, Medical Faculty Carl Gustav Carus, Technische Universitat Dresden, Dresden, Germany; Department of Medicine III, University Hospital Carl Gustav Carus, Dresden, Germany.
28
Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre, Madrid, Spain; Biomedical Research Networking Center on Rare Diseases (CIBERER), Madrid, Spain. Electronic address: mrobledo@cnio.es.

Abstract

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.

PMID:
28552549
PMCID:
PMC5500830
DOI:
10.1016/j.jmoldx.2017.04.009
[Indexed for MEDLINE]
Free PMC Article

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