Localization and characterization of genes involved in parathyroid tumor development

Sammanfattning: The overall goal of this thesis has been to identify new genes and characterize them regarding the involvement in parathyroid tumor development. The parathyroid glands are responsible for the control of calcium homeostasis. Parathyroid tumors usually have a benign growth pattern, with less than 1% of the tumors being malignant. The definition of hyperparathyroidism. (HPT) is that a patient has inappropriately high levels of parathyroid hormone (PTH) in relation to extracellular calcium. HPT is a common endocrinopathy and is generally caused by a monoclonal parathyroid adenoma. However, the etiologies behind the tumor development needs to be further elucidated. Secondary HPT (SHPT) arises in patients with renal failure, resulting in asymmetric hyperplasia of the parathyroid glands, but the mechanisms behind the tumorigenesis is unknown. A comparative genomic hybridization (CGH) analysis was performed in order to find numerical aberrations in SHPT tumors. Among 16 parathyroid glands from eight patients investigated, chromosomal abnormalities was detected in one gland only. The detected loss of chromosome 11 was coupled with a mutation in the MEN1 tumor suppressor gene in 11q13, shown to be responsible for some familial and sporadic forms of HPT. Hence, the majority of secondary HPT glands are chromosomally stable and can be expected to harbor more discrete genetic alterations, below the detection level of CGH (Paper I). We characterized the expression profiles in parathyroid adenomas and normal tissues, using the oligonucleotide microarray technology. Among the upregulated genes in adenomas vs. normal parathyroid, we identified several putative oncogenes implicated in cell growth and transcription regulation, such as the Cyclin D1 and the C-jun proto-oncogenes. Loss of chromosome 11 q is the best studied chromosomal alteration in parathyroid adenomas. Hence, we examined expression distinction between adenomas with or without 11 q loss. Unsupervised hierarchical clustering separated the tumor subsets well, and also the normal parathyroid samples (Paper II). Based on a positional candidate approach, the disease gene for the hyperparathyroidism -jaw tumor (HPT-JT) syndrome was isolated. Thirteen different heterozygous, germline, inactivating mutations in a single gene were identified in fourteen families with HPT-JT. The proposed role of HRPT2 (1q25) as a tumor suppressor was further supported from mutation screening in 48 parathyroid adenomas with cystic features, which revealed three somatic inactivating mutations. HRPT2 is a ubiquitously expressed, evolutionarily conserved gene, with a predicted protein of 531 amino acids, referred to as parafibromin (Paper III). The cellular function of Menin, the product of the MEN1 tumor suppressor gene, is still unknown. We have in mouse characterized the Men1 ortholog and identified a menin interacting partner. The Men1 gene was shown to be expressed in all organs investigated, at both RNA and protein levels. Four splice variants upstream of the translation initiation site were found and confirmed. The mRNA-ISH and Western blot analysis revealed Men1 RNA expression during the entire spermatogenesis. This suggests that menin may have a crucial function during the development of spermatids in agreement with the infertile phenotype of homozygous MEN1 humans. By a Yeast-two hybrid assay, the putative transcription factor gene pent, was identified and confirmed to be a menin interacting protein in mouse (Paper IV and V).