Analytical Performance of NGS-Based Molecular Genetic Tests Used in the Diagnostic Workflow of Pheochromocytoma/Paraganglioma.

Balazs Sarkadi,Gabor Nyiro,Henriett Butz,Henriett Butz,Istvan Liko,Peter Igaz,Attila Patocs

doi:10.3390/cancers13164219

Balazs Sarkadi, Gabor Nyiro + Show 5 more

Open Access

https://doi.org/10.3390/cancers13164219

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Journal: Cancers	Publication Date: Aug 22, 2021
Citations: 4	License type: CC BY 4.0

Affiliation: Semmelweis University, National Institute of Oncology

Abstract

Simple SummaryThe escalating use of Next Generation Sequencing in the routine clinical setting greatly facilitates the genetic diagnosis of hereditary cancer syndromes. However, these novel methods pose new and unique challenges. In our study we sought to demonstrate the evolution of these techniques, especially whole exome sequencing and targeted panel sequencing. This study highlights the multi-layered workflow and how each step affects the diagnostic outcome and demonstrates the effectiveness of an in-house developed targeted panel sequencing for hereditary endocrine tumor syndromes.Next Generation Sequencing (NGS)-based methods are high-throughput and cost-effective molecular genetic diagnostic tools. Targeted gene panel and whole exome sequencing (WES) are applied in clinical practice for assessing mutations of pheochromocytoma/paraganglioma (PPGL) associated genes, but the best strategy is debated. Germline mutations of at the least 18 PPGL genes are present in approximately 20–40% of patients, thus molecular genetic testing is recommended in all cases. We aimed to evaluate the analytical and clinical performances of NGS methods for mutation detection of PPGL-associated genes. WES (three different library preparation and bioinformatics workflows) and an in-house, hybridization based gene panel (endocrine-onco-gene-panel- ENDOGENE) was evaluated on 37 (20 WES and 17 ENDOGENE) samples with known variants. After optimization of the bioinformatic workflow, 61 additional samples were tested prospectively. All clinically relevant variants were validated with Sanger sequencing. Target capture of PPGL genes differed markedly between WES platforms and genes tested. All known variants were correctly identified by all methods, but methods of library preparations, sequencing platforms and bioinformatical settings significantly affected the diagnostic accuracy. The ENDOGENE panel identified several pathogenic mutations and unusual genotype–phenotype associations suggesting that the whole panel should be used for identification of genetic susceptibility of PPGL.

Highlights

Pheochromocytomas and paragangliomas (PPGL) are rare chromaffin cell tumors arising from the adrenal medulla or the sympathetic or parasympathetic paraganglia.PPGL have strong genetic determinism, overall approximately 40% of patients carry a germline mutation that predispose to the disease
The majority of these germline mutations occur in SDHB, SDHD, VHL, NF1, RET and KIF1B genes, but in rare or extremely rare cases, germline mutations of SDHA, SDHAF2, EGLN1, DLST, FH, MAX, MDH2, KMT2D, TMEM127, MERTK, MET and SLC25A11 genes [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23]
We summarize our experience with Next Generation Sequencing (NGS) based methods in molecular genetic testing of PPGL

Summary

Introduction

PPGL have strong genetic determinism, overall approximately 40% of patients carry a germline mutation that predispose to the disease The majority of these germline mutations occur in SDHB, SDHD, VHL, NF1, RET and KIF1B genes, but in rare or extremely rare cases, germline mutations of SDHA, SDHAF2, EGLN1, DLST, FH, MAX, MDH2, KMT2D, TMEM127, MERTK, MET and SLC25A11 genes [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23]. Not all mutations manifest with specific phenotype, and in some cases, due to the low number of documented patients, genotype–phenotype correlations are not yet established [1]

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