Markku Sainio

The ezrin protein family: membrane-cytoskeleton interactions and disease associations

Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. Ezrin protein subfamily members act as linkers between the plasma membrane and the cytoskeleton. Members of the subfamily have been shown to interact with each other, with cell adhesion molecules such as CD44 and with F-actin. Recent data indicate that intercellular adhesion molecules 1 and 2 also interact with ezrin. The proteins are also involved in the redistribution of intercellular adhesion molecules and the organization of cell membrane structures. Merlin is a tumor suppressor that is involved in tumorigenesis of schwannomas and meningiomas. Merlin has the same overall protein structure as the other proteins in the subfamily but may have partially distinct functions.

Long-Term Prognosis of Haemangioblastoma of the CNS: Impact of von Hippel-Lindau Disease

Summary¶ The aim was to assess the frequency of von Hippel-Lindau disease (VHL) and the long-term prognosis of VHL and non-VHL patients among 110 consecutive patients with haemangioblastoma (HB) of the CNS treated between 1953 and 1993 at one neurosurgical unit. To reveal VHL manifestations we performed a detailed clinical and radiological examination (neuraxis and abdomen) (61/110), VHL-gene mutation analysis (40/110), and collection of all available clinical, imaging, operative and autopsy data from the hospitals involved. All patients were followed-up with a median of 14 years (excluding 14 operative deaths), and no patient was lost to follow-up. Altogether 49 patients died during the follow-up. In the 14 VHL patients (13%), HB(s) of the CNS were detected at a median age of 33 years, retinal HB(s) at 39 years, and renal cell carcinoma (RCC) at 43 years. The frequency of VHL in patients operated on for HB(s) was 29% before the age of 25 years, 19% between 25 and 45 years, and only 2% after 45 years. HB patients not meeting the VHL criteria had internal organ cysts in 14%. One non-VHL patient (4%) had two adjacent HBs in the same cyst wall. The growth rates of non-VHL and VHL-related HBs were similar as indicated by the median time to recurrence and the proliferation indices (MIB-1). Recurrence of the HB in patients whose primary operation was considered radical developed in four of the 10 VHL patients at a median of 19 years, and in nine of the 74 non-VHL patients at a median of 11 years. The median length of life of all VHL and non-VHL patients was 46 and 63 years, respectively. In VHL, RCC and HBs were equal causes of death.

Structure-function relationships in the ezrin family and the effect of tumor-associated point mutations in neurofibromatosis 2 protein

Ezrin, radixin and moesin (ERM proteins) link cell adhesion molecules to the cytoskeleton, modulate cell morphology and cell growth and are involved in Rho-mediated signal transduction. Merlin, the tumor suppressor in neurofibromatosis 2, is a diverged member of the ezrin family, but its function is at least partially similar to the ERM proteins. In the N-domain, the ezrin family belongs to the band 4.1 superfamily. Secondary structure predictions made separately for the ezrin and band 4.1-tyrosine phosphatase families give a similar pattern for the homologous N-domains, indicating that both families have a similar binding site for the integral membrane proteins. The alpha-domain shows a strong coiled-coil prediction, that can be involved in the protein dimerization. The C-terminal actin-binding site in the ERM proteins and the actin-binding helix in the villin headpiece have a common amino acid motif. In merlin, the published tumor-associated single amino acid mutations in the N-domain are located in the conserved sites, and they affect mainly the predicted helices and strands, indicating that these mutations cause the disease primarily by disturbing the protein structure. In the alpha- and C-domains, some of the mutations break the helical structures. Some known mutations are observed at a site potentially interacting with cell adhesion molecules. We will also discuss the implications of the evolutionary information and the actin-binding models in the ezrin family.

Genetic aberrations in sporadic and neurofibromatosis 2 (NF2)-associated schwannomas studied by comparative genomic hybridization (CGH).

Summary¶ Background. Schwannomas occur sporadically or in association with neurofibromatosis 2 (NF2), an autosomal dominant disorder, which predisposes to multiple schwannomas, meningiomas and spinal ependymomas, with bilateral vestibular schwannomas as the classic hallmark. As NF2 and sporadic schwannomas differ in some respect in their clinical and biological behavior we evaluated whether there are any differences in the distribution of genetic aberrations between NF2 and sporadic schwannomas. Our interest was also to verify whether secondary genetic alterations besides the loss of 22q could be detected in schwannomas. Methods. We investigated DNA copy number changes in 25 schwannomas (12 NF2 and 13 sporadic schwannomas) using the comparative genomic hybridization (CGH) technique. Some chromosomal regions were further studied by LOH or FISH analysis. Findings. CGH detected genomic abnormalities in 15 of 25 schwannomas (60%). The most common alteration was loss on 22q, found in 32% (8/25) of schwannomas. No consistent changes were detected in other chromosomal regions. The overall number of genetic aberrations was similar in NF2 and in sporadic schwannomas. Interpretation. Our results support the present view that loss of chromosome 22q harboring the NF2 gene plays a universal role in the pathogenesis of schwannomas without consistent involvement of other chromosomal regions.

Proliferative potential of sporadic and neurofibromatosis 2-associated schwannomas as studied by MIB-1 (Ki-67) and PCNA labeling

Abstract. Neurofibromatosis 2 (NF2), a dominantly inherited disorder, is typically manifested as bilateral vestibular Schwannomas and predisposes to other nervous system tumors. Vestibular Schwannomas also occur sporadically but the onset is usually at an older age. Surgical and histological studies have shown that vestibular Schwannomas of NF2 patients are more invasive than sporadic Schwannomas and that the two groups also have morphological differences. We compared the proliferation activity of 26 vestibular Schwannomas (19 NF2 patients) to that of 27 sporadic cases using the Ki-67 (MIB-1) and PCNA (19A2) monoclonal antibodies. In addition, proliferation was assessed in 20 spinal benign Schwannomas, 4 spinal cellular Schwannomas and 3 spinal malignant peripheral nerve sheath tumors (MPNST). We found a significant difference in the proliferation potential between NF2 and sporadic vestibular Schwannomas (MIB-1-LI: 1.72 ± 0.93 vs 0.95 ± 0.57, p=0.001; and PCNA-LI: 1.40 ± 0.75 vs 0.81 ± 0.52, p=0.001). Age does not explain the detected difference in proliferation, since NF2 vestibular Schwannomas also had higher MIB-1 indices than 34 age-matched sporadic tumors. In spinal tumors, MPNST had higher MIB-1 indices than cellular Schwannomas, and therefore MIB-1 staining may be useful in distinguishing between them. Although the defective NF2 gene is important in the tumorigenesis of both NF2 and sporadic Schwannomas, our results suggest that there are differences in the molecular biology of these tumors.

Chromosome 22q alterations and expression of the NF2 gene product, merlin, in gastrointestinal stromal tumors

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the digestive tract. A characteristic genetic alteration in GISTs is constitutive activation of the c-kit proto-oncogene, but alterations in chromosomes 14 and 22 may also play a role in the molecular pathogenesis. In this study, 42 GISTs were analyzed for loss of heterozygosity (LOH) on the long arm of chromosome 22 (22q). Overall, 69% of the tumors studied showed LOH with at least 1 of the 22q markers. Allele losses were compared with tumor mitotic activity, the most commonly used prognostic marker for this tumor. Interestingly, allele deletion at 22q was significantly more frequent in tumors with high mitotic activity (>/= l2 mitoses/10 high-power fields [HPF]) than in tumors with low mitotic activity (< 2 mitoses/HPF)-88% versus 56% (P < 0.01). A total of 26% (11 of 42) of all tumors demonstrated loss of all 22q sites analyzed, consistent with the loss of 1 copy of the entire long arm. Such tumors carried a 4.6-fold (95% confidence interval, 0.5 to 49.8) risk for recurrence compared with tumors with no LOH. LOH was frequently detected at the neurofibromatosis 2 (NF2) tumor-suppressor gene locus at 22q12. Sequencing of the NF2 gene from 5 GISTs did not reveal mutations, however. Furthermore, 16 of 19 tumors (84%) analyzed by immunohistochemistry were positive for the NF2 gene product, merlin. The findings suggest that allelic losses at 22q are associated with high mitotic activity and recurring disease, and that alterations in the NF2 gene are unlikely to participate in the pathogenesis of GIST.

Recurrent DNA sequence copy losses on chromosomal arm 6q in capillary hemangioblastoma

Capillary hemangioblastomas (CHB) of the central nervous system, the most common tumor in von Hippel-Lindau (VHL) disease, usually show mutations in the VHL tumor suppressor gene on chromosome 3p25-p26. Because little is known concerning the cytogenetic changes in these tumors, we studied 22 cases through comparative genomic hybridization to screen for DNA copy number changes in both sporadic and VHL-associated CHB. Our analysis revealed that 6 of 22 samples (27%) contained DNA copy number losses, whereas no gains were observed. The most recurrent finding was loss of chromosomal arm 6q, seen in five cases. In two of these cases also loss of chromosome 3 was noted. The third aberration observed was loss of chromosome 8, seen in one case. No differences were noted between VHL-associated and sporadic tumors, nor did the cytogenetic aberrations correlate with the clinical outcome. The loss of 6q, seen in this study and previously in other VHL-associated tumors (renal cell carcinomas and pheochromocytomas) and other tumors, suggest that this chromosome area may contain tumor suppressor genes involved in the early steps of tumorigenesis.

Genomic structure of the human ezrin gene

The ERM proteins, ezrin, radixin, and moesin, act as linkers between the plasma membrane and actin cytoskeleton. They are involved in a variety of cellular functions, such as cell adhesion, migration, and the organization of cell surface structures, and are highly homologous, both in protein sequence and in functional activity, with merlin/schwannomin, a neurofibromatosis-2-associated tumor-suppressor protein. We report here the genomic structure and intron junction sequences of the human ezrin gene. Ezrin consists of 13 exons and spans approximately 24 kb genomic DNA. The coding parts of the exons range in size from 12 bp to 275 bp and the introns from 182 bp to 7 kb. The genomic structures of ezrin and moesin are highly conserved, suggesting their recent divergence. Radiation hybrid mapping has refined the location of ezrin to the interval between D6S442 and D6S281.