E. Montali

Screening for mutations in the neurofibromatosis type 2 (NF2) gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. They are usually sporadic but can also occur associated with the neurofibromatosis type 2 (NF2) syndrome. The gene responsible for NF2, recently isolated from chromosome 22, encodes a membrane-organizing protein that shows high sequence homology to a protein family thought to link the cytoskeleton with membrane proteins. Mutations of the NF2 gene have been described in sporadic meningiomas, exclusively in tumors that show loss of heterozygosity (LOH) of 22q. These preliminary results indicate that the NF2 gene is involved in the pathogenesis of at least a subset of meningiomas, where it does indeed behave as a tumor suppressor gene. In order to characterize better the role of the NF2 gene in the genesis of meningiomas we have examined the entire coding sequence of the gene in 125 meningiomas by single-strand conformational polymorphism analysis; furthermore, LOH analysis for markers of 22q has been carried out. Inactivating mutations were identified in 30% of our samples, all of which also showed LOH of 22q. The majority of mutations identified were frameshifts and nonsense mutations, which are predicted to produce a truncated or non-functional protein. We also found two missense and three in-frame deletions that may pinpoint specific regions of the protein critical to its function. Furthermore, the distribution of mutations throughout the gene, suggested that exons 2, 3, 5, 11 and 13 are more frequently involved. Our results reconfirm the importance of the NF2 gene in the pathogenesis of meningiomas and also suggest that there may be a nonrandom clustering of mutations throughout the gene.

Somatic mutations in the neurofibromatosis type 2 gene in sporadic meningiomas

Meningiomas are benign tumors of the central nervous system. Although usually sporadic, they can occur in patients affected by the autosomal dominant syndrome, neurofibromatosis type 2 (NF2). The NF2 gene has recently been isolated from chromosome 22. The presence of germline mutations in NF2 patients and the loss of heterozygosity (LOH) on 22q in NF2 tumors support the hypothesis that the NF2 gene acts as a tumor suppressor. Cytogenetic and LOH studies have suggested that the gene responsible for the development of meningiomas is located in the region of 22q in which the NF2 gene maps. The meningioma gene could therefore be the NF2 gene itself. Recently, somatic mutations of the NF2 gene have been identified in sporadic meningiomas, thus supporting the hypothesis that the NF2 gene is also important in meningioma pathogenesis. In this study, we analyzed sixty-one sporadic meningiomas for LOH of 22q and for mutations in the NF2 gene. LOH was detected in 36 of the 60 informative tumors. Single-strand conformational polymorphism analysis was used to identify nine mutations in five of the eight exons of the NF2 gene studied. The nine tumors with an altered NF2 gene also showed LOH for 22q markers. These results further support the hypothesis that mutations in the NF2 gene are a critical pathogenetic event in at least some meningiomas.

Prediction of recurrence by microsatellite analysis in head and neck cancer

We examined the possibility of using microsatellite alterations as markers to detect clonal tumor‐derived cell populations in histopathologically negative surgical margins and cervical lymph nodes from head and neck cancer (HNC) patients. We used polymerase chain reaction (PCR)‐based microsatellite analysis DNA to analyze primary tumors, paired surgical margins, and cervical lymph nodes from 41 HNC patients. Samples were scored for alterations as defined by the presence of new alleles (shifts) or loss of heterozygosity (LOH) at each of 10 markers. We identified 25 (61%) patients with primary HNC who appeared to have had a complete resection on the basis of the histopathological assessment and who were informative regarding microsatellite alterations in tumor tissue. In 11 of these 25 (44%) cases, PCR analysis of surgical margins showed the same microsatellite alterations as in the primary tumors. In 7 of these 11 patients, the carcinoma recurred locally, as compared with 1 out of 14 patients with negative margins (log rank test, P = 0.0049). Conversely, we were unable to detect clonal neoplastic cells in histopathologically negative lymph nodes examined by molecular analysis. Cox regression analysis showed that molecular positive margins were an independent prognostic factor (P = 0.04) for recurrence. This study demonstrates that microsatellite analysis may be a valuable tool for evaluating the risk of local recurrence.

Analysis of the neurofibromatosis type 2 gene in different human tumors of neuroectodermal origin

The autosomal dominant syndrome neurofibromatosis type 2 (NF2) is characterized by the development of bilateral vestibular schwannomas, meningiomas, ependymomas and gliomas. The NF2 gene, recently isolated from chromosome 22, is mutated in both sporadic and NF2 tumors such as schwannomas, meningiomas and ependymomas. Mutations of the gene have been described not only in the neoplasms usually associated with NF2, but also in 30% of the melanomas and 41 % of the mesotheliomas analyzed. In particular, the finding of mutations in melanomas supports the hypothesis that the NF2 gene is involved in the genesis of several tumor types that arise from the embryonic neural crest. In this study we examined, by single-strand conformation polymorphism (SSCP) analysis, 41 tumors of the central nervous system (11 schwannomas and 30 gliomas), 19 melanomas and 15 Merkel cell carcinoma specimens for mutations in the coding sequence of the NF2 gene. We found three inactivating mutations of the NF2 gene in schwannomas. No alterations of the gene were detected by SSCP analysis of the other tumors. These results confirm the role of NF2 in pathogenesis of schwannomas, but do not define its significance in the genesis of the other neuroectodermal tumors studied.

Evidence for a human mitotic mutant with pleiotropic effect

Male and female sibs born to third‐cousin parents presented with mental retardation, microcephaly, short stature, juvenile onset limb‐girdle muscular dystrophy and multiple chromosome mosaicism in lymphocytes and fibroblasts. Different aneuploidies (mostly trisomies) were found in 15–20% of the cells and trisomies for the chromosome 8 and chromosome 7 predominated in lymphocytes and fibroblasts respectively, while monosomies were rare. Increased cellular death due to aneuploidy could explain symptoms such as mental and growth retardation and microcephaly. This could be an instance of an autosomal recessive mitotic mutant, possibly affecting a protein simultaneously involved in spindle apparatus and muscle function.

Molecular Genetic Alterations of c-myc Oncogene in Superficial and Locally Advanced Bladder Cancer

Objective: Gene activation and altered expression of cellular proto-oncogene are important mechanisms implicated in initiation and development processes of human cancer. It has already been shown that c-myc oncogene is implicated in the control of cell proliferation, apoptosis and differentiation. Methods: We have determined the methylation status, the presence of genetic amplification and the presence of m-RNA overexpression of c-myc gene in 31 samples from patients with bladder carcinomas. Results: Our data demonstrated the presence of c-myc gene amplification only in 5 of 15 superficial bladder carcinomas (p < 0.05). On the other hand, we did not find statistical significant correlation between the methylation, expression of c-myc gene and the clinical-histopathological parameters. A significant correlation (p < 0.05) was found between the methylation pattern and m-RNA overexpression of c-myc oncogene. Conclusion: We demonstrate aberrant c-myc gene status in human bladder cancer. This oncogene is altered at different levels in bladder carcinoma genesis and progression.

Analysis of two 47,XXX males reveals X-Y interchange and maternal or paternal nondisjunction

SummaryTwo cases of 47,XXX males were studied, one of which has been published previously (Bigozzi et al. 1980). Analysis of X-linked restriction fragment length polymorphisms revealed that in this case, one X chromosome was of paternal and two were of maternal origin, whereas in the other case, two X chromosomes were of paternal and one of maternal origin. Southern blot analysis with Y-specific DNA probes demonstrated the presence of Y short arm sequences in both XXX males. In one case, the results obtained pointed to a paracentric inversion on Yp of the patients father. In situ hybridization indicated that the Y-specific DNA sequences were localized on Xp22.3 in one of the three X chromosomes in both cases. The presence of Y DNA had no effect on random X inactivation. It is concluded that both XXX males originate from aberrant X-Y interchange during paternal meiosis, with coincident nondisjunction of the X chromosome during maternal meiosis in case 1, and during paternal meiosis II in case 2.

Abnormal c-myc oncogene DNA methylation in human bladder cancer : Possible role in tumor progression

OBJECTIVE It has been suggested that the hypermethylation of normally unmethylated DNA sequences plays a critical role in the genesis and progression of human tumors. Although the molecular bases of this mechanism have not been completely explained, the altered methylation pattern of the c-myc oncogene is supposed to represent an important step in tumor development. METHODS We have analyzed tissue samples from 47 urinary bladder tumors (43 primary transitional and 4 squamous cell carcinomas) and the respective blood with HpaII methyl-sensitive endonuclease digestion and the Southern blotting technique to detect the methylation pattern in a widespread area in and around the c-myc oncogene. RESULTS Data presented in this study showed significant differences between the c-myc methylation pattern and pathological grade (p < 0.05). On the other hand, we did not find a significant correlation between the c-myc methylation pattern and clinical stage. However, a variable covalent alteration of c-myc DNA existed in bladder cancer as compared to normal tissue. CONCLUSION Although the correlation between superficial and infiltrating forms was not statistically significant, we did, however, find differences in aggressive neoplastic behavior. This suggested that local hypermethylation may be considered as one potential mechanism for increasing genetic alterations in bladder cancer formation.

Morbilità e Mortalità nella Prole di 300 Coppie di Coniugi Consanguinei nel Comune di Firenze

A family investigation has been carried out on 301 consanguineous and the same number of nonconsanguineous couples, who married in Florence in the years 1939 through 1958. The sample turned out to be homogeneous as regards age of the couple, year of marriage, period of cohabitation, social level, and methods of survey. The average rate of consanguinity in Florence in the above years was of 0.458 × 10 −3 , with a decrease from 0.595 to 0.327 × 10 −3 between the first and the second decade. While the number of pregnancies is not significantly different in the two groups, abortions are more frequent among blood relatives. As a result, the number of stillbirths being not significantly different, the birth rate is higher among the controls. Infant mortality is more than double in blood relatives. As regards morbidity, hereditary defects or anomalies show an average increase of 2.6 times in the offspring of blood relatives: the increase being higher for serious hereditary defects (3.44) than for lighter ones (1.98). The finding of slight delays in mental development was 4.8 times more frequent in the offspring of blood relatives. By applying Morton-Crow-Mullers formula, it was possible to calculate the A and B values, and to find out, in the observed population, the presence of 2.2-2.4 pathological gene equivalents per gamete as to early mortality (abortions + stillbirths + infant mortality); 1.6-1.7 gene equivalents as to hereditary defects in general; and 0.82-0.85 gene equivalents as to slight delays in mental development.

In vitro expression of proα1(I) collagen mRNA by human pre-osteoclastic cells

In vitro studies have demonstrated that the extracellular matrix modulates the cell phenotype. In the present study we have investigated in vitro proα1(I) collagen mRNA expression in a human pre-osteoclastic cell line (FLG 29.1 cells) in basal condition and after various stimuli. In addition, in order to evaluate the effect of cell-cell interactions on collagen type I mRNA expression, we have cultured the human pre-osteoclastic cells FLG 29.1 with either the human osteoblast-like cell line Saos-2 or the bovine bone endothelial cell line BBE. We showed that the FLG 29.1 cells express proα1(I) collagen mRNA, whose expression is modulated by phorbol esters (TPA). Co-culturing FLG 29.1 cells with either Saos-2 or BBE cells induced decrease of proα1(I) collagen mRNA expression.