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Genetic Aspects of Endocrine Tumorigenesis: A Hunt for the Endocrine Neoplasia Gene
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Endocrine Surgery. (Experimental surgery)
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 [en]
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: urn:nbn:se:uu:diva-224111ISBN: 978-91-554-8973-1 (print)OAI: oai:DiVA.org:uu-224111DiVA: diva2:715344
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
List of papers
1. Expression and somatic mutations of SDHAF2 (SDH5), a novel endocrine tumor suppressor gene in parathyroid tumors of primary hyperparathyroidism
Open this publication in new window or tab >>Expression and somatic mutations of SDHAF2 (SDH5), a novel endocrine tumor suppressor gene in parathyroid tumors of primary hyperparathyroidism
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2010 (English)In: Endocrine, ISSN 0969-711X, Vol. 38, no 3, 397-401 p.Article in journal (Refereed) Published
Abstract [en]

To investigate the SDHAF2 gene and its effect on primary hyperparathyroidism. Parathyroid tumors causing primary hyperparathyroidism (pHPT) are one of the more common endocrine neoplasias. Loss of heterozygosity at chromosome 11q13 is the most common chromosomal aberration in parathyroid tumors occurring in about 40% of sporadic tumors. Only 15-19% display somatic mutations in the MEN1 gene, which suggest that this chromosomal region may harbor additional genes of importance in parathyroid tumor development. The SDHAF2 (formerly SDH5) gene is a recently identified neuroendocrine tumor suppressor gene at this locus, and inherited mutations of the SDHAF2 gene has been linked to familial paraganglioma. We demonstrate that the SDHAF2 gene is expressed in parathyroid tissue using RT-PCR. Because detection of inactivating mutations is the major criterion for validating a candidate tumor suppressor, we used automated sequencing of the coding region and intron/exon boundaries in 80 sporadic parathyroid adenomas from patients with pHPT. A known polymorphisms (A to G substitution; rs879647) was identified in 9/80 parathyroid tumors but no tumor-specific somatic mutational aberrations, such as nonsense, frameshift, or other inactivating mutations were identified. The SDHAF2 gene is expressed in parathyroid tissue. However, somatic mutations of the SDHAF2 tumor suppressor gene are unlikely to frequently contribute to parathyroid tumor development in sporadic pHPT.

Keyword
Hyperparathyroidism, Parathyroid, Paraganglioma, Succinate dehydrogenase, SDHAF2
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-139273 (URN)10.1007/s12020-010-9399-0 (DOI)000284116000010 ()
Available from: 2010-12-22 Created: 2010-12-22 Last updated: 2016-12-20Bibliographically approved
2. Epigenetic inactivation of SDHAF2 is a frequent event in benign and malignant pheochromocytomas.
Open this publication in new window or tab >>Epigenetic inactivation of SDHAF2 is a frequent event in benign and malignant pheochromocytomas.
(English)Manuscript (preprint) (Other academic)
National Category
Clinical Medicine Basic Medicine
Identifiers
urn:nbn:se:uu:diva-224109 (URN)
Available from: 2014-05-04 Created: 2014-05-04 Last updated: 2014-06-30
3. Global DNA methylation patterns in small intestinal neuroendocrine tumors (SI-NETs)
Open this publication in new window or tab >>Global DNA methylation patterns in small intestinal neuroendocrine tumors (SI-NETs)
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2014 (English)In: Endocrine-Related Cancer, ISSN 1351-0088, E-ISSN 1479-6821, Vol. 21, no 1, L5-L7 p.Article in journal (Refereed) Published
Abstract [en]

Small intestinal neuroendocrine tumors (SI-NETs) are rare hormone producing tumors and are often diagnosed at advanced stage. The genetic and epigenetic background of SI-NETs are poorly understood, but several reports have indicated chromosomal losses at 18.21-qter and 11q22-q23. The aim of this study was to characterize CpG DNA methylation status of primary SI-NETs and the corresponding lymph node metastases. We used the commercially available HumanMethylation27 Beadchip array (Illumina), which covers 27578 CpG sites spanning over 14495 genes, and analyzed a discovery cohort of 10 primary SI-NETs with matched metastases. Messenger- mRNA, were determined for selected genes in a 47 tumors. In comparison to the primary tumors, the metastases showed 2697 statistically significant differentially genes. Metastases were generally less methylated than primary tumors. The relative mRNA expression level of the differentially methylated genes AXL, CRMP1, FGF5, and APOBEC3C largely reflected the methylation status. MAPK4, RUNX3, TP73, CCND1, CHFR, AHRR, and Rb1 known to be hypermethylated in other cancer types, displayed overall high methylation level (β-value ≥ 0.9). Methylation (β -value >0,7) at 18q21-qter and 11q22-q23 were detected in genes SETBP1, ELAC1, MBD1, MAPK4, TCEB3C and ARVC1, MMP8, BTG4, APOA1, FAM89B, HSPB1, respectively. Furthermore unsupervised clustering of the tumors identified three distinct clusters, one with a highly malignant behavior. Our data supports involvement of CpG DNA methylation in metastatic progression of SI-NETs and this could present a possibility to identify more aggressive tumors based on DNA methylation.

National Category
Surgery
Identifiers
urn:nbn:se:uu:diva-212142 (URN)10.1530/ERC-13-0481 (DOI)000334279600002 ()24192231 (PubMedID)
Available from: 2013-12-06 Created: 2013-12-06 Last updated: 2017-12-06
4. Exome Sequencing reveal no recurrent mutations on chromosome 18 in small intestinal neuroendocrine tumors; Ruling out a suspect?
Open this publication in new window or tab >>Exome Sequencing reveal no recurrent mutations on chromosome 18 in small intestinal neuroendocrine tumors; Ruling out a suspect?
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(English)Manuscript (preprint) (Other academic)
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-224110 (URN)
Available from: 2014-05-04 Created: 2014-05-04 Last updated: 2014-06-30
5. 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
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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.

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

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