Molecular profiling related to poor prognosis in thyroid carcinoma. Combining gene expression data and biological information.

Undifferentiated and poorly differentiated thyroid tumors are responsible for more than half of thyroid cancer patient deaths in spite of their low incidence. Conventional treatments do not obtain substantial benefits, and the lack of alternative approaches limits patient survival. Additionally, the absence of prognostic markers for well-differentiated tumors complicates patient-specific treatments and favors the progression of recurrent forms. In order to recognize the molecular basis involved in tumor dedifferentiation and identify potential markers for thyroid cancer prognosis prediction, we analysed the expression profile of 44 thyroid primary tumors with different degrees of dedifferentiation and aggressiveness using cDNA microarrays. Transcriptome comparison of dedifferentiated and well-differentiated thyroid tumors identified 1031 genes with >2-fold difference in absolute values and false discovery rate of <0.15. According to known molecular interaction and reaction networks, the products of these genes were mainly clustered in the MAPkinase signaling pathway, the TGF-beta signaling pathway, focal adhesion and cell motility, activation of actin polymerization and cell cycle. An exhaustive search in several databases allowed us to identify various members of the matrix metalloproteinase, melanoma antigen A and collagen gene families within the upregulated gene set. We also identified a prognosis classifier comprising just 30 transcripts with an overall accuracy of 95%. These findings may clarify the molecular mechanisms involved in thyroid tumor dedifferentiation and provide a potential prognosis predictor as well as targets for new therapies.

Role of apoA-II in lipid metabolism and atherosclerosis: advances in the study of an enigmatic protein.

Our understanding of apolipoprotein A-II (apoA-II) physiology is much more limited than that of apoA-I. However, important and rather surprising advances have been produced, mainly through analysis of genetically modified mice. These results reveal a positive association of apoA-II with FFA and VLDL triglyceride plasma concentrations; however, whether this is due to increased VLDL synthesis or to decreased VLDL catabolism remains a matter of controversy. As apoA-II-deficient mice present a phenotype of insulin hypersensitivity, a function of apoA-II in regulating FFA metabolism seems likely. Studies of human beings have shown the apoA-II locus to be a determinant of FFA plasma levels, and several genome-wide searches of different populations with type 2 diabetes have found linkage to an apoA-II intragenic marker, making apoA-II an attractive candidate gene for this disease. The increased concentration of apoB-containing lipoproteins present in apoA-II transgenic mice explains, in part, why these animals present increased atherosclerosis susceptibility. In addition, apoA-II transgenic mice also present impairment of two major HDL antiatherogenic functions: reverse cholesterol transport and protection of LDL oxidative modification. The apoA-II locus has also been suggested as an important genetic determinant of HDL cholesterol concentration, even though there is a major species-specific difference between the effects of mouse and human apoA-II. As antagonizing apoA-I antiatherogenic actions can hardly be considered the apoA-II function in HDL, this remains a topic for future investigations. We suggest that the existence of apoA-II or apoA-I in HDL could be an important signal for specific interaction with HDL receptors such as cubilin or heat shock protein 60.

Genetic, clinical, and biochemical analysis of unrelated Spanish families with multiple endocrine neoplasia type I.

OBJECTIVE: (1) To study seven unrelated Spanish families with multiple endocrine neoplasia type I (MEN I), describing clinical features and investigating the presence of germline mutations in the MEN1 gene, and (2) to establish reference values for pancreatic polypeptide and gastrin after a standardized test meal in a healthy control group, analyzing the usefulness of this test for detecting neuroendocrine gastroenteropancreatic tumors in subjects with MEN I. METHODS: Two or three generations of 7 kindreds with MEN I, consisting of a total of 39 individual family members, were investigated. Three of the families were subjected only to genetic analysis, and the other four families were also assessed clinically. A group of 23 healthy control subjects were also studied. RESULTS: Mutations in the MEN1 gene were found in six of the seven families studied. Of the 4 families studied clinically, 12 family members were genetically affected. In these study subjects, hyperparathyroidism, adrenal adenomas, neuroendocrine gastroenteropancreatic tumors, and pituitary adenomas developed in 100%, 50%, 16%, and 12%, respectively. All demonstrated pancreatic tumors were associated with abnormal results after a test meal, but 75% of them also showed high basal hormonal measurements. CONCLUSION: Analysis of the MEN1 gene decreases the total number of subjects who need to undergo repeated clinical and biochemical studies, but genetic mutations are not detected in all families with MEN I. Hyperparathyroidism is the most common manifestation of the syndrome, but the presence of adrenal adenomas has probably been underestimated. Ingestion of a standardized test meal for stimulation of gastrin and pancreatic polypeptide could be a complementary procedure for diagnosing gastroenteropancreatic tumors in selected patients with MEN I in whom basal gastrin and pancreatic polypeptide levels are normal.

Molecular pathology of multiple endocrine neoplasia type I: two novel germline mutations and updated classification of mutations affecting MEN1 gene.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by the combined development of tumors in several endocrine glands and other tissues. The MEN1 gene was recently identified and isolated by positional cloning. This gene was screened in two unrelated MEN1 Spanish kindreds (with four affected members and seven asymptomatic members) using single-strand conformation polymorphism, DNA sequencing, and restriction enzyme analysis. Two novel germline mutations were identified: a missense in exon 2 (H139R) and a splice-site in intron 9 (1461-2A>C). These findings allowed us to identify the MEN1 carriers among the seven asymptomatic members analyzed. An updated review of the mutations and polymorphisms found in the analysis of the MEN1 gene is provided. The report of all germline mutations causing MEN1 and easy access to this updated information are both of special diagnostic interest, because this greatly facilitates the task of attributing the disorder to a specific mutation found in a given MEN1 family. This is especially helpful in the critical differentiation of missense mutations from nonsynonymous polymorphisms that fit the pattern of segregation of the disease, but do not cause it.

A novel germline mutation in exon 5 of the multiple endocrine neoplasia type 1 gene.

The autosomal dominant multiple endocrine neoplasia type 1 (MEN1) syndrome is characterized by neoplasia of parathyroids, anterior pituitary, and gastrointestinal and pancreatic neuroendocrine tissues. Recently the gene responsible for the MEN1 syndrome has been identified on chromosome region 11q13. Most of the described mutations are nucleotide substitutions and small deletions affecting exons 2 and 3, causing protein truncation. Only one mutation in exon 5 has been found, and this corresponds to a MEN1 sporadic case. Small insertions are also rare. We studied a MENI family composed of five members, two of whom were clinically affected. We found a new germline 1 basepair insertional mutation affecting the exon 5 of the MEN1 gene in the two members affected in this MEN1 family.

Molecular diagnosis of lecithin: cholesterol acyltransferase deficiency in a presymptomatic proband.

We report the molecular diagnosis of a lecithin : cholesterol acyltransferase deficiency in a 12-year old proband with a high-density lipoprotein deficiency. The increased percentage of free cholesterol in plasma and high-density lipoprotein indicated an inherited lecithin : cholesterol acyltransferase deficiency as the underlying cause. This diagnosis was confirmed by a low plasma lecithin : cholesterol acyltransferase activity and a combination of genetic analyses which demonstrated compound heterozygosity for two mutations in the lecithin : cholesterol acyltransferase gene of the proband. One was a previously unreported 2 bp deletion leading to a stop signal in codon 77 and the other a point mutation causing Arg 135-->Gln transition. To our knowledge, this is the first diagnosis of lecithin : cholesterol acyltransferase deficiency in a pre-symptomatic patient. Whether the proband will develop signs of complete lecithin : cholesterol acyltransferase deficiency or the milder form (Fish Eye Disease) is uncertain, although the former possibility is more likely. The risk of premature atherosclerosis conferred by lecithin : cholesterol acyltransferase deficiency is not well established. The proband will need to be carefully monitored in the future.

Undetected viral infection in diffuse alveolar damage associated with bone marrow transplantation.

To investigate the presence of nondiagnosed viral lung infections in patients who developed acute respiratory failure and diffuse pulmonary infiltrates after bone marrow transplantation (BMT), we studied necropsy-obtained lung specimens with features of diffuse alveolar damage (DAD) where no other specific histological diagnosis could be established, by using in situ hybridization and immunohistochemistry. Lung tissue samples obtained at necropsy from 19 patients (12 males and 7 females; 31 +/- 11 yrs mean +/- SD age) who died 56 +/- 36 days after BMT (12 allogeneic and 7 autologous), were studied retrospectively using specific deoxyribonucleic acid (DNA) probes to detect cytomegalovirus (CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV), and adenovirus genomes. Tissue samples were additionally processed with antibodies to CMV and HSV antigens. Cells infected by CMV were detected by in situ hybridization in five cases, and by immunohistochemistry in four cases. Combining the results of both procedures, a previously undiagnosed CMV infection was found in six patients. All of them had received an allogeneic BMT and had developed graft-versus-host disease (GVHD). No evidence of cells infected by HSV, EBV, or adenovirus was found in any case. No viral infection was detected either in recipients of autologous marrow or in recipients of allogeneic BMT without GVHD. These results indicate that pulmonary cytomegalovirus infection not detected by conventional histological examinations may be present in patients with diffuse alveolar damage associated with bone marrow transplantation, especially in recipients of allogeneic marrow who develop graft-versus-host disease. Furthermore, the use of in situ hybridization and/or immunohistochemistry on pulmonary histology might improve the diagnosis of viral lung infections in patients receiving bone marrow transplantation.

Molecular characterization of the breakpoints of an inversion fixed between Drosophila melanogaster and D. subobscura.

The two breakpoints of a chromosomal inversion fixed since the split of Drosophila melanogaster and D. subobscura lineages have been isolated and sequenced in both species. The regions spanning the breakpoints initially were identified by the presence of two signals after interspecific in situ hybridization on polytene chromosomes. Interspecific comparison of the sequenced regions allowed us to delineate the location of the breakpoints. Close to one of these breakpoints a new transcription unit (bcn92) has been identified in both species. The inversion fixed between D. melanogaster and D. subobscura does not seem to have broken any transcription unit. Neither complete nor defective transposable elements were found in the regions encompassing the breakpoints. Short thymine-rich sequences (30-50 bp long) have been found bordering the breakpoint regions. Although alternating Pur-Pyr sequences were detected, these putative target sites for topoisomerase II were not differentially clustered in the breakpoints.

Intraspecific and interspecific variation at the y-ac-sc region of Drosophila simulans and Drosophila melanogaster.

A 2.2-kb region including the ac gene of Drosophila simulans has been sequenced. Interspecific divergence between Drosophila melanogaster and D. simulans was estimated as 0.0695 and 0.0558 for silent and for all sites, respectively. Estimated silent site divergence for the ac region is comparable to that estimated for other regions of the genome between these species, indicating that silent sites of the ac region are not under significantly stronger functional constraint. Intraspecific variation in both species was also investigated. Restriction-site and length polymorphism in the ac region of D. simulans has been investigated for 103 X chromosome lines sampled from three natural populations in Spain using eight four-cutter restriction enzymes. Neither restriction-site nor length variation was detected in the three populations surveyed. In D. melanogaster restriction-site and length polymorphism in all major transcription units of the y-ac-sc region (23.1-kb region) has been studied using four four-cutter restriction enzymes for 245 X chromosome lines sampled from 10 natural populations (seven from Europe, two from North America and one from Japan). Fourteen restriction-site and 28 length polymorphisms were detected. There was some indication of population subdivision for North American vs. European samples of D. melanogaster. The frequency spectrum of restriction-site polymorphisms in European populations was skewed toward rarer frequencies than predicted by the neutral theory. Comparison of silent site variation at this telomeric region with that in the Adh 5'-flanking region showed a reduced level of heterozygosity in the y-ac-sc region. Since interspecific silent divergence is not reduced in the y-ac-sc region as compared to other regions, the reduction in standing levels of variation at this telomeric locus in both D. simulans and D. melanogaster is most easily explained by a hitchhiking effect of linked selected substitutions.