Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Charting the Genetic Landscape and Clonal Architectures of Pheochromocytoma
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences. (Experimentell Kirurgi)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Genotypic and phenotypic inter patient heterogeneity characterize pheochromocytoma and paraganglioma (PPGL). Up to 60% of PPGL are associated with either somatic or germline mutations in at least 14 established disease causing genes. Consequently, a comprehensive screening test for PPGL patients utilizing standard techniques is not feasible and in the diagnostic approach, multiple different phenotype guided gene prioritization protocols have been utilized. This may result in misdiagnosis, especially in patients with sporadic presentation. Diagnostic testing of somatic mutations in tumour material is not performed due to the lack of actionable results.

The aims of this study were, (1) to investigate the use of novel sequencing techniques in a clinical application, (2) to discover novel PPGL disease causing loci using novel sequencing techniques, (3) to characterize a large cohort of PPGL for mutations in known disease causing genes and to analyse corresponding genotype-phenotype correlations, (4) to dissect the molecular and genetic landscape of MEN2 PPGL and (5) to determine the clonal architecture and heterogeneity within, and in-between matched PPGL.

For these purposes we studied PPGL tumours from a total of 96 patients using targeted and/or whole exome enrichment, capillary and high throughput sequencing as well as genome wide array based genotyping. Novel bioinformatics pipelines were constructed for raw data processing and downstream interpretation. Quantitative PCR, western blot and immunohistochemistry were utilized in order to characterize molecular traits. Selected experimental findings were correlated to patient phenotype.

We conclude that novel sequencing techniques could be utilized in clinical genetic screening of patients with PPGL. Somatic gain-of-function mutations in H-RAS are likely to contribute to disease pathogenesis. Analysing tumour DNA for somatic mutations in disease causing genes could provide relevant clinical information and have an impact on patient management. Concomitant mutations in PPGL may occur in exceptional cases and have a substantial impact on tumour biology and patient phenotype. And finally genetic heterogeneity is present between and within a majority of PPGL tumours.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. , 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1047
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:uu:diva-234285ISBN: 978-91-554-9084-3 (print)OAI: oai:DiVA.org:uu-234285DiVA: diva2:757063
Public defence
2014-12-06, Auditorium Minus, Gustavianum, Akademigatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2014-11-13 Created: 2014-10-15 Last updated: 2015-02-03
List of papers
1. Next-generation sequencing in the clinical genetic screening of patients with pheochromocytoma and paraganglioma
Open this publication in new window or tab >>Next-generation sequencing in the clinical genetic screening of patients with pheochromocytoma and paraganglioma
Show others...
2013 (English)In: Endocrine connections, ISSN 2049-3614, Vol. 2, no 2, 104-111 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND:

Recent findings have shown that up to 60% of pheochromocytomas (PCCs) and paragangliomas (PGLs) are caused by germline or somatic mutations in one of the 11 hitherto known susceptibility genes: SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, HIF2A (EPAS1), RET, NF1, TMEM127 and MAX. This list of genes is constantly growing and the 11 genes together consist of 144 exons. A genetic screening test is extensively time consuming and expensive. Hence, we introduce next-generation sequencing (NGS) as a time-efficient and cost-effective alternative.

METHODS:

Tumour lesions from three patients with apparently sporadic PCC were subjected to whole exome sequencing utilizing Agilent Sureselect target enrichment system and Illumina Hi seq platform. Bioinformatics analysis was performed in-house using commercially available software. Variants in PCC and PGL susceptibility genes were identified.

RESULTS:

We have identified 16 unique genetic variants in PCC susceptibility loci in three different PCC, spending less than a 30-min hands-on, in-house time. Two patients had one unique variant each that was classified as probably and possibly pathogenic: NF1 Arg304Ter and RET Tyr791Phe. The RET variant was verified by Sanger sequencing.

CONCLUSIONS:

NGS can serve as a fast and cost-effective method in the clinical genetic screening of PCC. The bioinformatics analysis may be performed without expert skills. We identified process optimization, characterization of unknown variants and determination of additive effects of multiple variants as key issues to be addressed by future studies.

National Category
Surgery
Identifiers
urn:nbn:se:uu:diva-212875 (URN)10.1530/EC-13-0009 (DOI)23781326 (PubMedID)
Available from: 2013-12-16 Created: 2013-12-16 Last updated: 2016-12-21Bibliographically approved
2. Somatic Mutations in H-RAS in Sporadic Pheochromocytoma and Paraganglioma Identified by Exome Sequencing
Open this publication in new window or tab >>Somatic Mutations in H-RAS in Sporadic Pheochromocytoma and Paraganglioma Identified by Exome Sequencing
Show others...
2013 (English)In: Journal of Clinical Endocrinology and Metabolism, ISSN 0021-972X, E-ISSN 1945-7197, Vol. 98, no 7, E1266-E1271 p.Article in journal (Refereed) Published
Abstract [en]

Context: Up to 60% of pheochromocytoma (PCC) and paraganglioma (PGL) are associated with either somatic or germline mutations in established PCC and PGL susceptibility loci. Most unexplained cases are characterized by an increased activity of the RAS/RAF/ERK signaling pathway. Mutations in RAS subtypes H, K, and N are common in human cancers; however, previous studies have been inconsistent regarding the mutational status of RAS in PCC and PGL. Objectives: The aim of this study was to identify novel disease causing genes in PCC and PGL tumors. Design, setting, and participants: Four benign and sporadic PCC and PGL tumors were subjected to whole exome sequencing using the Illumina HiSeq Platform. Sequences were processed by CLC genomics 4.9 bioinformatics software and the acquired list of genetic variants was filtered against the Catalogue of Somatic Mutations in Cancer database. Findings were validated in an additional 78 PCC and PGL tumor lesions. Results: Exome sequencing identified 2 cases with somatic mutations in the H-RAS. In total, 6.9% (n = 4/58) of tumors negative for mutations in major PCC and PGL loci had mutations in H-RAS: G13R, Q61K, and Q61R. There were 3 PCC and 1 PGL; all had sporadic presentation with benign tumor characteristics and substantial increases in norepinephrine and/or epinephrine. H-RAS tumors were exclusively found in male patients (P = .007). Conclusions: We identified recurrent somatic H-RAS mutations in pheochromocytoma and paraganglioma. Tumors with H-RAS mutations had activation of the RAS/RAF/ERK signaling pathway and were associated with male PCC patients having benign and sporadic disease characteristics. H-RAS could serve as a prognostic and predictive marker as well as a novel therapeutic target.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-208673 (URN)10.1210/jc.2012-4257 (DOI)000322780600017 ()
Available from: 2013-10-07 Created: 2013-10-07 Last updated: 2017-12-06Bibliographically approved
3. Integrative Genetic Characterization and Phenotype Correlations in Pheochromocytoma and Paraganglioma Tumours
Open this publication in new window or tab >>Integrative Genetic Characterization and Phenotype Correlations in Pheochromocytoma and Paraganglioma Tumours
Show others...
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 1, e86756- p.Article in journal (Refereed) Published
Abstract [en]

Background: About 60% of Pheochromocytoma (PCC) and Paraganglioma (PGL) patients have either germline or somatic mutations in one of the 12 proposed disease causing genes; SDHA, SDHB, SDHC, SDHD, SDHAF2, VHL, EPAS1, RET, NF1, TMEM127, MAX and H-RAS. Selective screening for germline mutations is routinely performed in clinical management of these diseases. Testing for somatic alterations is not performed on a regular basis because of limitations in interpreting the results. Aim: The purpose of the study was to investigate genetic events and phenotype correlations in a large cohort of PCC and PGL tumours. Methods: A total of 101 tumours from 89 patients with PCC and PGL were re-sequenced for a panel of 10 disease causing genes using automated Sanger sequencing. Selected samples were analysed with Multiplex Ligation-dependent Probe Amplification and/or SNParray. Results: Pathogenic genetic variants were found in tumours from 33 individual patients (37%), 14 (16%) were discovered in constitutional DNA and 16 (18%) were confirmed as somatic. Loss of heterozygosity (LOH) was observed in 1/1 SDHB, 11/11 VHL and 3/3 NF1-associated tumours. In patients with somatic mutations there were no recurrences in contrast to carriers of germline mutations (P = 0.022). SDHx/VHL/ EPAS1 associated cases had higher norepinephrine output (P = 0.03) and lower epinephrine output (P<0.001) compared to RET/NF1/H-RAS cases. Conclusion: Somatic mutations are frequent events in PCC and PGL tumours. Tumour genotype may be further investigated as prognostic factors in these diseases. Growing evidence suggest that analysis of tumour DNA could have an impact on the management of these patients.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-220300 (URN)10.1371/journal.pone.0086756 (DOI)000330283100207 ()
Available from: 2014-03-17 Created: 2014-03-12 Last updated: 2017-12-05Bibliographically approved
4. Concurrent somatic VHL deletion and point mutation in a Multiple Endocrine 2 Neoplasia type  metastatic pheochromocytoma.
Open this publication in new window or tab >>Concurrent somatic VHL deletion and point mutation in a Multiple Endocrine 2 Neoplasia type  metastatic pheochromocytoma.
(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-234284 (URN)
Available from: 2014-10-15 Created: 2014-10-15 Last updated: 2015-02-03
5. Spatio-temporal heterogeneity characterize the genetic landscape of pheochromocytoma and defines early events in tumorigenesis.
Open this publication in new window or tab >>Spatio-temporal heterogeneity characterize the genetic landscape of pheochromocytoma and defines early events in tumorigenesis.
(English)Manuscript (preprint) (Other academic)
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-234283 (URN)
Available from: 2014-10-15 Created: 2014-10-15 Last updated: 2015-02-03

Open Access in DiVA

fulltext(1364 kB)451 downloads
File information
File name FULLTEXT01.pdfFile size 1364 kBChecksum SHA-512
ec2cba101b9e54aa6d7edaebd9ad196c6cd75c0eeffbee2210d66e37a7472b1069f80dbd8ffff817e6cd14c52139de18790aecde97dd9869bab6b9f8414ce8ce
Type fulltextMimetype application/pdf
Buy this publication >>

Search in DiVA

By author/editor
Crona, Joakim
By organisation
Department of Surgical Sciences
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar
Total: 451 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1634 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf