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New Insights in Genetic and Epigenetic Mechanisms Involved in Parathyroid Tumorigenesis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences. (Experimental Surgery)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Primary hyperparathyroidism (pHPT) is a pathology associated with one or multiple hyperfunctioning parathyroid glands.  The disease prevalence occurs in roughly 1-2% of the population primarily post-menopausal women.  The molecular pathology of the disease is poorly understood.  Elevated serum calcium levels in the setting of an inappropriately elevated parathyroid hormone level are indicative of the disease process.  The ultimate treatment of the disease is to remove the hyperfunctioning gland.

The aim of this thesis was to examine potential genetic and epigenetic aberrations that are potentially disease causing.

The methylation signature of normal and pathological parathyroid tissue has yet to be investigated.  DNA was bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident.  DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 known to be important in the development of parathyroid tumors were associated with reduced gene expression in both benign and malignant parathyroid tumors.

Familial primary hyperparathyroidism (FPHPT) may occur due to an underlying germ-line mutation in the MEN1, CASR, or HRPT2/CDC73 genes.  Eighty-six young (45 years of age) patients with clinically non-syndromic PHPT underwent genetic analysis.  Eight of 86 (9.3%) young patients with clinically non-familial PHPT displayed deleterious germ-line mutations in the susceptibility genes (4 MEN1, 3 CASR, and 1 HRPT2/ CDC73).

Accumulation of non-phosphorylated active β -catenin has been reported to commonly occur in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT).  We assessed possible β-catenin stabilizing mutations in a large series of parathyroid adenomas. A total of one hundred and eighty sporadic parathyroid adenomas were examined for mutations in exon 3 of the CTNNB1gene. The mutation S33C (TCT >TGT) was detected by direct-DNA sequencing of PCR fragments in 1 out of 180 sporadic parathyroid adenomas (0.68 %).

Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing.  Four of eight tumors displayed a frame shift deletion or nonsense mutations within the MEN1 gene, which was accompanied by loss of heterozygosity of the remaining wild-type allele.  One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%).

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 36 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 927
Keyword [en]
Parathyroid, yumorigenesis, mutation, exome sequencing
National Category
Medical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-205587ISBN: 978-91-554-8727-0 (print)OAI: oai:DiVA.org:uu-205587DiVA: diva2:642110
Public defence
2013-10-03, Rosen salen, Akademiska sjukhuset, ingång 95, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-09-10 Created: 2013-08-20 Last updated: 2014-01-22
List of papers
1. The DNA Methylome of Benign and Malignant Parathyroid Tumors
Open this publication in new window or tab >>The DNA Methylome of Benign and Malignant Parathyroid Tumors
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2011 (English)In: Genes, Chromosomes and Cancer, ISSN 1045-2257, E-ISSN 1098-2264, Vol. 50, no 9, 735-745 p.Article in journal (Refereed) Published
Abstract [en]

The role of DNA methylation of CpG islands in parathyroid tumorigenesis has not been analyzed in an unbiased, systematic fashion. DNA was isolated from normal and pathologic parathyroid tissues, bisulphite modified and analyzed using the Infinium HumanMethylation27 BeadChip. Distinct hierarchical clustering of genes with altered DNA methylation profiles in normal and pathologic parathyroid tissue was evident. Comparing normal parathyroid tissue with parathyroid adenomas, 367 genes were significantly altered, while 175 genes significantly differed when comparing parathyroid carcinomas and normal parathyroid tissues. A comparison between parathyroid adenomas and parathyroid carcinomas identified 263 genes with significantly distinct methylation levels. Results were confirmed for certain genes in a validation cohort of 40 parathyroid adenomas by methylation-specific PCR. Genes of known or putative importance in the development of parathyroid tumors showed significant and frequent hypermethylation. DNA hypermethylation of CDKN2B, CDKN2A, WT1, SFRP1, SFRP2, and SFRP4 was associated with reduced gene expression in both benign and malignant parathyroid tumors. Treatment with 5-aza-2 '-deoxycytidine of primary cell cultures restores expression of hypermethylated genes in benign and malignant parathyroid tumors. In conclusion, the unbiased, genome-wide study of the parathyroid tumor DNA methylome identified a number of genes with altered DNA methylation patterns of putative importance to benign and malignant parathyroid tumorigenesis.

National Category
Surgery
Identifiers
urn:nbn:se:uu:diva-156583 (URN)10.1002/gcc.20895 (DOI)000292702900007 ()
Available from: 2011-08-07 Created: 2011-08-04 Last updated: 2017-12-08
2. Frequent germ-line mutations of the MEN1, CASR, and HRPT2/CDC73 genes in young patients with clinically non-familial primary hyperparathyroidism
Open this publication in new window or tab >>Frequent germ-line mutations of the MEN1, CASR, and HRPT2/CDC73 genes in young patients with clinically non-familial primary hyperparathyroidism
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2012 (English)In: Hormones & Cancer, ISSN 1868-8497, Vol. 3, no 1-2, 44-51 p.Article in journal (Refereed) Published
Abstract [en]

Familial primary hyperparathyroidism (FPHPT) may occur due to an underlying germ-line mutation in the MEN1, CASR, or HRPT2/CDC73 genes. The disease may be undiagnosed in the absence of a history suggestive of FHPT. Young PHPT patients (≤45 years of age) are more likely to harbor occult FPHPT. A total of 1,161 (136 were ≤45 years of age) PHPT patients underwent parathyroidectomy from 2001 to 2009. Thirty-four patients declined participation. Sixteen patients were diagnosed in the clinical routine with FPHPT (11 MEN1, four MEN2A, and one HPT-JT) and were not included in the genetic analysis. Eighty-six young (≤45 years of age) patients with clinically non-syndromic PHPT underwent genetic analysis. Sanger sequencing of all coding regions of the MEN1, CASR, and the HRPT2/CDC73 genes was performed. Eight of 86 (9.3%) young patients with clinically non-familial PHPT displayed deleterious germ-line mutations in the susceptibility genes (4 MEN1, 3 CASR, and 1 HRPT2/CDC73). There was one insertion, one deletion, two nonsense, and four missense mutations, all predicted to be highly damaging to protein function and absent in 3,244 control chromosomes. Germ-line mutations in known susceptibility genes within young patients with PHPT, including those diagnosed in the clinical routine, was 24/102 (23.5%; 15 MEN1, four RET, three CASR, and two HRPT2/CDC73). We demonstrate that germ-line inactivating mutations in susceptibility genes are common in young patients with clinically non-familial PHPT. Thus, enhanced use of genetic analysis may be warranted in clinically non-familial young PHPT patients.

National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-205584 (URN)10.1007/s12672-011-0100-8 (DOI)22187299 (PubMedID)
Available from: 2013-08-20 Created: 2013-08-20 Last updated: 2016-12-21Bibliographically approved
3. Evidence of a stabilizing mutation of beta-catenin encoded by CTNNB1 exon 3 in a large series of sporadic parathyroid adenomas
Open this publication in new window or tab >>Evidence of a stabilizing mutation of beta-catenin encoded by CTNNB1 exon 3 in a large series of sporadic parathyroid adenomas
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2012 (English)In: Endocrine (Basingstoke), ISSN 1355-008X, E-ISSN 1559-0100, Vol. 42, no 3, 612-615 p.Article in journal (Refereed) Published
Abstract [en]

Aberrant accumulation of beta-catenin plays an important role in a variety of human neoplasms. This can be caused by stabilizing mutation of beta-catenin (CTNNB1, exon 3) or by mutation or deregulated expression of other components of the WNT/beta-catenin signaling pathway. Accumulation of non-phosphorylated active beta-catenin has been reported to commonly occur in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT), either due to the aberrantly spliced internally truncated WNT receptor LRP5 (LRP5 Delta) or to a stabilizing mutation of beta-catenin. The S37A mutation was reported to occur in 7.3 % in a single study of parathyroid adenomas, while in other studies no stabilizing mutations of beta-catenin exon 3 were identified. The aim of this study was to determine the mutational frequency of the CTNNB1 gene, specifically exon 3 in a large series of parathyroid adenomas. One hundred and eighty sporadic parathyroid adenomas were examined for mutations in exon 3 of CTNNB1 by direct DNA sequencing, utilizing previously published primer sequences. The mutation S33C (TCT > TGT) was detected by direct-DNA sequencing of PCR fragments in 1 out of 180 sporadic parathyroid adenomas (0.68 %). Like serine 37, mutations of serine 33 have been reported in many neoplasms with resulting beta-catenin stabilization, enhanced transcription, and oncogenic activities. Immunohistochemical analysis revealed an overexpression of the beta-catenin protein in the lone mutant tumor. Taking also previous studies into account we conclude that activating mutations of the regulatory GSK-3 beta phosphorylation sites serine 33 and 37, encoded by CTNNB1 exon 3, rarely occur in parathyroid adenomas from patients with pHPT.

Keyword
Parathyroid, Primary hyperparathyroidism, Beta-catenin
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-191036 (URN)10.1007/s12020-012-9690-3 (DOI)000312073500023 ()
Note

Correction in: Endocrine, vol. 42, issue 3, pg 769

DOI: 10.1007/s12020-012-9763-3

Available from: 2013-01-09 Created: 2013-01-09 Last updated: 2017-12-06
4. Identification of Somatic Mutations in Parathyroid Tumors Using Whole-Exome Sequencing
Open this publication in new window or tab >>Identification of Somatic Mutations in Parathyroid Tumors Using Whole-Exome Sequencing
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2012 (English)In: Journal of Clinical Endocrinology and Metabolism, ISSN 0021-972X, E-ISSN 1945-7197, Vol. 97, no 9, E1774-E1781 p.Article in journal (Refereed) Published
Abstract [en]

Context: The underlying molecular alterations causing sporadic parathyroid adenomas that drive primary hyperparathyroidism have not been thoroughly defined.

Objective: The aim of the study was to investigate the occurrence of somatic mutations driving tumor formation and progression in sporadic parathyroid adenoma using whole-exome sequencing.

Design: Eight matched tumor-constitutional DNA pairs from patients with sporadic parathyroid adenomas underwent whole-exome capture and high-throughput sequencing. Selected genes were analyzed for mutations in an additional 185 parathyroid adenomas.

Results: Four of eight tumors displayed a frame shift deletion or nonsense mutation in MEN1, which was accompanied by loss of heterozygosity of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N mutation of the histone methyltransferase EZH2 gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in the additional 185 parathyroid adenomas revealed a high rate of MEN1 mutations (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic EZH2 mutation that was found by exome sequencing.

Conclusion: This study confirms the frequent role of the loss of heterozygosity of chromosome 11 and MEN1 gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, EZH2, in endocrine tumorigenesis.

National Category
Clinical Medicine
Identifiers
urn:nbn:se:uu:diva-183226 (URN)10.1210/jc.2012-1743 (DOI)000308692900020 ()22740705 (PubMedID)
Available from: 2012-10-25 Created: 2012-10-23 Last updated: 2017-12-07Bibliographically approved

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