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Next-generation sequencing in the clinical genetic screening of patients with pheochromocytoma and paraganglioma
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Experimental Surgery.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Experimental Surgery.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Endocrine Oncology.
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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.

Place, publisher, year, edition, pages
2013. Vol. 2, no 2, 104-111 p.
National Category
Surgery
Identifiers
URN: urn:nbn:se:uu:diva-212875DOI: 10.1530/EC-13-0009PubMedID: 23781326OAI: oai:DiVA.org:uu-212875DiVA: diva2:679405
Available from: 2013-12-16 Created: 2013-12-16 Last updated: 2016-12-21Bibliographically approved
In thesis
1. Genetic Aspects of Endocrine Tumorigenesis: A Hunt for the Endocrine Neoplasia Gene
Open this publication in new window or tab >>Genetic Aspects of Endocrine Tumorigenesis: A Hunt for the Endocrine Neoplasia Gene
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Endocrine tumors arise from endocrine glands. Most endocrine tumors are benign but malignant variants exist. Several endocrine neoplasms display loss of parts of chromosome 11 or 18, produce hormones and responds poorly to conventional chemotherapeutics. The multiple endocrine neoplasia syndromes are mainly confined to endocrine tumors. This opens the question if there exists a single or several endocrine tumor genes.

The aim of the study was to describe genetic derangements in endocrine tumors.

Paper I: Investigation of mutational status of SDHAF2 in parathyroid tumors. SDHAF2 is located in the proximity of 11q13, a region that frequently displays loss in parathyroid tumors. We established that mutations in SDHAF2 are infrequent in parathyroid tumors.

Paper II: Study of SDHAF2 gene expression in a cohort of benign pheochromocytomas (PCC) (n=40) and malignant PCC (n=10). We discovered a subset of  benign PCC (28/40) and all malignant PCC (10/10) with significantly lower SDHAF2 expression. Benign PCC with low SDHAF2 expression and malignant tumors consistently expressing low levels of SDHAF2 were methylated in the promoter region. SDHAF2 expression was restored in vitro after treatment with 5- aza-2-deoxycytidine.

Paper III: HumanMethylation27 array (Illumina) covering 27578 CpG sites spanning over 14495 genes were analyzed in a discovery cohort of 10 primary small neuroendocrine tumors (SI-NETs) with matched metastases. 2697 genes showed different methylation pattern between the primary tumor and its metastasis. We identified several hypermethylated genes in key regions. Unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior.

Paper IV: Loss of chromosome 18 is the most frequent genetic aberration in SI-NETs. DNA from SI-NETs were subjected to whole exome capture sequencing and high resolution SNP array. Genomic profiling revealed loss of chromosome 18 in 5 out of 7 SI-NETs. No tumor-specific somatic mutation on chromosome 18 was identified which suggests involvement of other mechanisms than point mutations in SI-NET tumorigenesis.

Paper V: The cost for diagnostic genetic screening of common susceptibility genes in PCC is expensive and labor intensive. Three PCC from three patients with no known family history were chosen for exome capture sequencing. We identified three variants in known candidate genes. We suggest that exome-capture sequencing is a quick and cost-effective tool.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1010
Keyword
Exome sequencing, SDHAF2, epigenetics, methylation, methylation array, Sanger sequencing, pheochromocytoma, SI-NETs, carcinoid, oncology, endocrine surgery, parathyroid
National Category
Surgery Medical Genetics
Research subject
Genetics; Surgery
Identifiers
urn:nbn:se:uu:diva-224111 (URN)978-91-554-8973-1 (ISBN)
Public defence
2014-08-29, Grönwallsalen, Ing. 70, Akademiska Sjukhuset, Uppsala, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2014-06-05 Created: 2014-05-04 Last updated: 2014-06-30Bibliographically approved
2. Charting the Genetic Landscape and Clonal Architectures of Pheochromocytoma
Open this publication in new window or tab >>Charting the Genetic Landscape and Clonal Architectures of Pheochromocytoma
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:nbn:se:uu:diva-234285 (URN)978-91-554-9084-3 (ISBN)
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

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Crona, JoakimVerdugo, Alberto DelgadoGranberg, DanWelin, StaffanStålberg, PeterHellman, PerBjörklund, Peyman

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