<p> Tumorigenesis of relatively late onset occurring in patients with no family history of cancer syndromes is assumed to be driven by somatic mutations. The advent of high-throughput sequencing allows unbiased probing for genomic aberrations on an unprecedented scale. Somatic mutations, insertions and deletions, and copy number variations are able to be identified by parallel sequencing of tumor DNA and normal DNA from an individual patient. Somatic aberrations identified are classified as either passenger mutations that do not contribute to tumorigenesis or pathogenic driver mutations. Driver mutations are able to be identified due to their recurrence across multiple affected patients at a frequency greater than would be expected by chance.</p><p> Tumors occurring in the same tissue and from the same cell type often display diverse phenotypes with distinct mutational signatures. Therefore I applied high-throughput sequencing to probe for somatic mutations in two very specific endocrine tumor types - parathyroid-producing adenomas and insulin-producing adenomas (insulinomas). Prior to this study, neither tumor type had been probed for somatic mutations in a large-scale, unbiased manner. Though a limited number of mutated genes had been identified to play a role in familial and sporadic tumorigenesis in these tumor types, the majority of pathogenesis remained unexplained.</p><p> In order to maximize detection of variation in coding regions of the genome, an exome capture array was applied to the DNA prior to sequencing. In both tumor types, exome sequencing was applied to a small number of tumor-normal tissue pairs. Additional targeted sequencing of candidate driver mutations was then performed using Sanger sequencing on larger validation cohorts of tumors.</p><p> Exome sequencing revealed few somatic, protein-altering mutations in each tumor type (average <4 per tumor), therefore any recurrent variation was highly probable to be tumorigenic. Exome sequencing of the parathyroid adenomas revealed that four of eight tumors harbored a frameshift deletion or nonsense mutation in <i>MEN1</i>, which was always accompanied by loss of heterozygosity (LOH) of the remaining wild-type allele. No other mutated genes were shared among the eight tumors. One tumor harbored a Y641N missense mutation of the histone methyltransferase <i>EZH2</i> gene, previously linked to myeloid and lymphoid malignancy formation. Targeted sequencing in an additional 185 parathyroid adenomas revealed somatic <i>MEN1</i> mutations in a large number of tumors (35%). Furthermore, this targeted sequencing identified an additional parathyroid adenoma that contained the identical, somatic <i>EZH2</i> mutation that was found by exome sequencing. This confirms the frequent role of LOH of chromosome 11 and <i>MEN1</i> gene alterations in sporadic parathyroid adenomas and implicates a previously unassociated methyltransferase gene, <i>EZH2</i>, in endocrine tumorigenesis. </p><p> Exome sequencing identified an identical somatic, heterozygous mutation in Yin Yang 1 transcription factor (<i>YY1</i>) in two of seven insulinomas. Targeted sequencing of an additional 36 insulinomas revealed twelve more insulinomas that harbored this identical T372R missense mutation in <i>YY1.</i> This mutation occurs at a highly-conserved residue in a highly-conserved zinc finger DNA-binding domain. ChIP-Seq demonstrated that this mutation changes the DNA motif bound by YY1. This altered binding likely drives pathogenesis due to aberrant regulation of genes not regulated by YY1<sup>WT</sup>. With the goal of identifying differentially-expressed genes in <i>YY1<sup>T372R</sup></i> tumors, I performed gene expression analysis on eleven tumors; six that were <i>YY1<sup>WT</sup></i> and five that were <i>YY1<sup>T372R.</sup></i> This demonstrated that YY1<sup>T372R</sup> imparts a unique expression signature. Interestingly, several differentially-expressed genes were involved in key pathways regulating insulin secretion, including <i>ADCY1</i> (an adenylyl cyclase) and <i>CACNA2D2</i> (a Ca<sup>2+</sup> channel pore-forming subunit), both of which were upregulated in <i>YY1<sup>T372R</sup></i>-tumors. Importantly, <i>in vitro</i> studies using the INS-1 rat insulinoma cell line demonstrated that upregulation of each of gene is sufficient to markedly increase insulin secretion. Furthermore, both genes harbored specific YY1<sup>T372R</sup> binding sites that may account for their significantly altered expression.</p><p> Both studies identify novel driver mutations that shed light on the mechanisms of endocrine tumorigenesis. Furthermore, my findings reinforce the notion that common somatic mutations within the exome account for the majority of instances of sporadic tumorigenesis.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:3580657 |
| Date | 26 June 2014 |
| Creators | Cromer, Michael Kyle |
| Publisher | Yale University |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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