Molecular and Clinical Characteristics of Pituitary Adenoma Predisposition (PAP)

Pituitary adenomas are common benign neoplasms. Although most of them are sporadic, a minority occurs in familial settings. Heterozygous germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene were found to underlie familial pituitary adenomas, a condition designated as pituitary adenoma predisposition (PAP). PAP confers incomplete penetrance of mostly growth hormone (GH) secreting adenomas in young patients, who often lack a family history of pituitary adenomas. This thesis work aimed to clarify the molecular and clinical characteristics of PAP. Applying the multiplex ligation-dependent probe amplification assay (MLPA), we found large genomic AIP deletions to account for a subset of PAP. Therefore, MLPA could be considered in PAP suspected patients with no AIP mutations found with conventional sequencing. We generated an Aip mouse model to examine pituitary tumorigenesis in vivo. The heterozygous Aip mutation conferred complete penetrance of pituitary adenomas that were mostly GH-secreting, rendering the phenotype of the Aip mouse similar to that of PAP patients. We suggest that AIP may function as a candidate gatekeeper gene in somatotrophs. To clarify molecular mechanisms of tumorigenesis, we elucidated the expression of AIP-related molecules in human and mouse pituitary tumors. The expression of aryl hydrocarbon receptor nuclear translocator (ARNT) was reduced in mouse Aip-deficient adenomas, and similar ARNT reduction was also evident in human AIP mutation positive adenomas. This suggests that in addition to participating in the hypoxia pathway, estrogen receptor signaling and xenobiotic response pathways, ARNT may play a role in AIP-related tumorigenesis. We also studied the characteristics and the response to therapy of PAP patients and found them to have an aggressive disease phenotype with young age at onset. Therefore, improvement in treatment outcomes of PAP patients would require their efficient identification and earlier diagnosis of the pituitary adenomas. The possible role of the RET proto-oncogene in tumorigenesis of familial AIP mutation negative pituitary adenomas was evaluated, but none of the found RET germline variants were considered pathogenic. Surprisingly, RET immunohistochemistry suggested possible underexpression of RET in AIP mutation positive pituitary adenomas an observation that merits further investigation.

Molecular features of colorectal cancer predisposition and progression

Both inherited genetic variations and somatically acquired mutations drive cancer development. The aim of this thesis was to gain insight into the molecular mechanisms underlying colorectal cancer (CRC) predisposition and tumor progression. Whereas one-third of CRC may develop in the context of hereditary predisposition, the known highly penetrant syndromes only explain a small fraction of all cases. Genome-wide association studies have shown that ten common single nucleotide polymorphisms (SNPs) modestly predispose to CRC. Our population-based sample series of around thousand CRC cases and healthy controls was genotyped for these SNPs. Tumors of heterozygous patients were analyzed for allelic imbalance, in an attempt to reveal the role of these SNPs in somatic tumor progression. The risk allele of rs6983267 at 8q24 was favored in the tumors significantly more often than the neutral allele, indicating that this germline variant is somatically selected for. No imbalance targeting the risk allele was observed in the remaining loci, suggesting that most of the low-penetrance CRC SNPs mainly play a role in the early stages of the neoplastic process. The ten SNPs were further analyzed in 788 CRC cases, 97 of which had a family history of CRC, to evaluate their combined contribution. A significant association appeared between the overall number of risk alleles and familial CRC and these ten SNPs seem to explain around 9% of the familial clustering of CRC. Finding more CRC susceptibility alleles may facilitate individualized risk prediction and cancer prevention in the future. Microsatellite instability (MSI), resulting from defective mismatch repair function, is a hallmark of Lynch syndrome and observed in a subset of all CRCs. Our aim was to identify microsatellite frameshift mutations that inactivate tumor suppressor genes in MSI CRCs. By sequencing microsatellite repeats of underexpressed genes we found six novel MSI target genes that were frequently mutated in 100 MSI CRCs: 51% in GLYR1, 47% in ABCC5, 43% in WDTC1, 33% in ROCK1, 30% in OR51E2, and 28% in TCEB3. Immunohistochemical staining of GLYR1 revealed defective protein expression in homozygously mutated tumors, providing further support for the loss of function hypothesis. Another mutation screening effort sought to identify MSI target genes with putative oncogenic functions. Microsatellites were similarly sequenced in genes that were overexpressed and, upon mutation, predicted to avoid nonsense-mediated mRNA decay. The mitotic checkpoint kinase TTK harbored protein-elongating mutations in 59% of MSI CRCs and the mutant protein was detected in heterozygous MSI CRC cells. No checkpoint dysregulation or defective protein localization was observable however, and the biological relevance of this mutation may hence be related to other mechanisms. In conclusion, these two large-scale and unbiased efforts identified frequently mutated genes that are likely to contribute to the development of this cancer type and may be utilized in developing diagnostic and therapeutic applications.

Molecular diagnosis of developmental disorders

ABSTRACT Idiopathic developmental disorders (DDs) affect ~1% of the population worldwide. This being a considerable amount, efforts are being made to elucidate the disease mechanisms. One or several genetic factors cause 30-40% of DDs, and only 10% are caused by environmental factors. The remaining 50% of DD patients go undiagnosed, mostly due to a lack of diagnostic techniques. The cause in most undiagnosed cases is though to be a genetic factor or a combination of genetic and environmental factors. Despite the surge of new technologies entering the market, their implementation into diagnostic laboratories is hampered by costs, lack of information about the expected diagnostic yield, and the wide range of selection. This study evaluates new microarray methods in diagnosing idiopathic DDs, providing information about their added diagnostic value. Study I analysed 150 patients by array comparative genomic hybridization (array CGH, 44K and 244K), with a subsequent 18% diagnostic yield. These results are supported by other studies, indicating an enourmous added diagnostic value of array CGH, compared with conventional cytogenetic analysis. Nevertheless, 80% of the patients remained undiagnosed in Study I. In an effort to diagnose more patients, in Study IV the resolution was increased from 8.9 Kb of the 244K CGH array to 0.7 Kb, by using a single-nucleotide polymorphism (SNP) array. However, no additional pathogenic changes were detected in the 35 patients assessed, and thus, for diagnostic purposes, an array platform with ca 9 Kb resolution appears adequate. The recent vast increase in reports of detected aberrations and associated phenotypes has enabled characterization of several new syndromes first based on a common aberration and thereafter by delineation of common clinical characteristics. In Study II, a familial deletion at 9q22.2q22.32 with variable penetrance was described. Despite several reports of aberrations in the adjacent area at 9q associated with Gorlin syndrome, the patients in this family had a unique phenotype and did not present with the syndrome. In Study III, a familial duplication of chromosome 6p22.2 was described. The duplication caused increased expression of an important enzyme of the γ-aminobutyric acid (GABA) degradation pathway, causing oxidative stress of the brain, and thus, very likely, the mild mental retardation of these patients. These two case studies attempted to pinpoint candidate genes and to resolve the pathogenic mechanism causing the clinical characteristics of the patients. Presenting rare genetic and clinical findings to the international science and medical community enables interpretation of similar findings in other patients. The added value of molecular karyotyping in patients with idiopathic DD is evident. As a first line of testing, arrays with a median resolution of at least 9 Kb should be considered and further characterization of detected aberrations undertaken when possible. Diagnostic whole-exome sequencing may be the best option for patients who remain undiagnosed after high-resolution array analysis.