Neurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be... Show moreNeurofibromatosis type 1 (NF1) is caused by inactivating mutations in NF1. Due to the size, complexity, and high mutation rate at the NF1 locus, the identification of causative variants can be challenging. To obtain a molecular diagnosis in 15 individuals meeting diagnostic criteria for NF1, we performed transcriptome analysis (RNA-seq) on RNA obtained from cultured skin fibroblasts. In each case, routine molecular DNA diagnostics had failed to identify a disease-causing variant in NF1. A pathogenic variant or abnormal mRNA splicing was identified in 13 cases: 6 deep intronic variants and 2 transposon insertions causing noncanonical splicing, 3 postzygotic changes, 1 branch point mutation and, in 1 case, abnormal splicing for which the responsible DNA change remains to be identified. These findings helped resolve the molecular findings for an additional 17 individuals in multiple families with NF1, demonstrating the utility of skin-fibroblast-based transcriptome analysis for molecular diagnostics. RNA-seq improves mutation detection in NF1 and provides a powerful complementary approach to DNA-based methods. Importantly, our approach is applicable to other genetic disorders, particularly those caused by a wide variety of variants in a limited number of genes and specifically for individuals in whom routine molecular DNA diagnostics did not identify the causative variant. Show less
The neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has... Show moreThe neuroendocrine tumours paraganglioma and pheochromocytoma (PPGLs) are commonly associated with succinate dehydrogenase (SDH) gene variants, but no human SDH-related PPGL-derived cell line has been developed to date. The aim of this study was to systematically explore practical issues related to the classical 2D-culture of SDH-related human paragangliomas and pheochromocytomas, with the ultimate goal of identifying a viable tumour-derived cell line. PPGL tumour tissue/cells (chromaffin cells) were cultured in a variety of media formulations and supplements. Tumour explants and dissociated primary tumour cells were cultured and stained with a range of antibodies to identify markers suitable for use in human PPGL culture. We cultured 62 PPGLs, including tumours with confirmed SDHB, SDHC and SDHD variants, as well as several metastatic tumours. Testing a wide range of basic cell culture media and supplements, we noted a marked decline in chromaffin cell numbers over a 4-8 week period but the persistence of small numbers of synaptophysin/tyrosine hydroxylase-positive chromaffin cells for up to 99 weeks. In cell culture, immunohistochemical staining for chromogranin A and neuron-specific enolase was generally negative in chromaffin cells, while staining for synaptophysin and tyrosine hydroxylase was generally positive. GFAP showed the most consistent staining of type II sustentacular cells. Of the media tested, low serum or serum-free media best sustained relative chromaffin cell numbers, while lactate enhanced the survival of synaptophysin-positive cells. Synaptophysin-positive PPGL tumour cells persist in culture for long periods but show little evidence of proliferation. Synaptophysin was the most consistent cell marker for chromaffin cells and GFAP the best marker for sustentacular cells in human PPGL cultures. Show less
Infantile fibrosarcoma (IFS) and congenital mesoblastic nephroma (CMN) are locally aggressive tumors primarily occurring in infants. Both IFS and the cellular subtype of CMN show overlapping... Show moreInfantile fibrosarcoma (IFS) and congenital mesoblastic nephroma (CMN) are locally aggressive tumors primarily occurring in infants. Both IFS and the cellular subtype of CMN show overlapping morphological features and an ETV6-NTRK3 fusion, suggesting a close relationship. An activating alteration of EGFR, based on an EGFR kinase domain duplication (KDD), occurs in a subset of CMNs lacking an NTRK3 rearrangement, especially in the classic and mixed type. So far no EGFR-KDDs have been detected in IFS.We describe four pediatric tumors at the extremities (leg, n = 2; foot and arm n = 1) with histological features of IFS/CMN. Two cases showed classic IFS morphology while two were similar to classic/mixed type CMN. In all cases, an EGFR-KDD was identified without detection of a fusion gene. There were no abnormalities of the kidneys in any of the patients.This is the first description of IFS with an EGFR-KDD as driver mutation, supporting that IFS and CMN are similar lesions with the same morphological and genetic spectrum. Pathologists should be aware of the more fibrous variant of IFS, similar to classic/mixed type CMN. Molecular analyses are crucial to treat these lesions adequately, especially with regard to the administration of tyrosine kinase inhibitors. Show less
Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications)... Show moreNext-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity. Show less
Purpose: Paragangliomas of the head and neck are neuroendocrine tumors and are associated with germ line mutations of the tricarboxylic acid cycle-related genes SDHB, SDHC, SDHD, and SDHAF2.... Show morePurpose: Paragangliomas of the head and neck are neuroendocrine tumors and are associated with germ line mutations of the tricarboxylic acid cycle-related genes SDHB, SDHC, SDHD, and SDHAF2. Hypoxia is important in most solid tumors, and was directly implicated in tumorigenesis over 40 years ago when it was shown that dwelling at high altitudes increases the incidence of carotid body hyperplasia and paragangliomas. Although recent research has now elucidated several pathways of hypoxia in paragangliomas, nothing is currently known of the genetics or of gene-environment interactions in high-altitude paraganglioma. We postulated that SDH mutations might play a role in these tumors. Experimental Design: Patients from a Mexican family, originating and resident in Guadalajara, were tested for mutations of SDHD, and subsequently, for mutations of SDHB followed by immunohistochemical confirmation of SDHB loss. Results: Two patients, born and resident at altitudes of between 1,560 and 2,240 m, were found to have head and neck paragangliomas, including a remarkably aggressive recurrent tumor. Mutation analysis identified a pathogenic missense mutation in exon 7 of SDHB, c. 689G>A, p.Arg230His, and loss of the SDHB protein was confirmed by immunohistochemistry. Conclusions: This is the first report of a SDH gene mutation in paraganglioma at high altitude. A rapidly recurrent head and neck paraganglioma is a very rare finding in an SDH mutation carrier, suggesting a gene-environment interaction. Neither patient showed evidence of sympathetic paraganglioma. Clin Cancer Res; 16(16); 4148-54. (C) 2010 AACR. Show less
Background Paragangliomas and phaeochromocytomas are neuroendocrine tumours associated frequently with germline mutations of SDHD, SDHC, and SDHB. Previous studies have shown the imprinted SDHAF2... Show moreBackground Paragangliomas and phaeochromocytomas are neuroendocrine tumours associated frequently with germline mutations of SDHD, SDHC, and SDHB. Previous studies have shown the imprinted SDHAF2 gene to be mutated in a large Dutch kindred with paragangliomas. We aimed to identify SDHAF2 mutation carriers, assess the clinical genetic significance of SDHAF2, and describe the associated clinical phenotype. Methods We undertook a multicentre study in Spain and the Netherlands in 443 apparently sporadic patients with paragangliomas and phaeochromocytomas who did not have mutations in SDHD, SDHC, or SDHB. We analysed DNA of 315 patients for germline mutations of SDHAF2; a subset (n=200) was investigated for gross gene deletions. DNA from a group of 128 tumours was studied for somatic mutations. We also examined a Spanish family with head and neck paragangliomas with a young age of onset for the presence of SDHAF2 mutations, undertook haplotype analysis in this kindred, and assessed their clinical phenotype. Findings We did not identify any germline or somatic mutations of SDHAF2, and no gross gene deletions were noted in the subset of apparently sporadic patients analysed. Investigation of the Spanish family identified a pathogenic germline DNA mutation of SDHAF2, 232G -> A (Gly78Arg), identical to the Dutch kindred. Interpretation SDHAF2 mutations do not have an important role in phaeochromocytoma and are rare in head and neck paraganglioma. Identification of a second family with the Gly78Arg mutation suggests that this is a crucial residue for the function of SDHAF2. We conclude that SDHAF2 mutation analysis is justified in very young patients with isolated head and neck paraganglioma without mutations in SDHD, SDHC, or SDHB, and in individuals with familial antecedents who are negative for mutations in all other risk genes. Show less
