Gene-Targeted Analysis of Clinically Diagnosed Long QT Russian Families.

Long QT syndrome (LQTS) has great genetic heterogeneity: more than 500 mutations have been described in several genes. Despite many advances, a genetic diagnosis still cannot be established in 25-30% of patients. The aim of the present study was to perform genetic evaluation in 9 Russian families with LQTS; here we report the results of 4 positive probands and their relatives (a total of 16 individuals). All subjects underwent clinical examination, 12-lead ECG, and Holter monitoring. Genetic analysis of the 14 genes mainly involved in LQTS was performed using a next-generation sequencing approach. We identified two new mutations (KCNQ1 gene) and 6 known mutations (AKAP9, ANK2, KCNE1 and KCNJ2 genes) in 4 out of 9 probands, some of which have already been described in association with LQTS. Segregation studies suggest a possible causative role for KCNQ1 p.(Leu342Pro), AKAP9 p.(Arg1609Lys), KCNE1 p.(Asp85Asn), and KCNJ2 p.(Arg82Gln) variations. Our study confirmed the high genetic heterogeneity of this disease and highlights the difficulties to reveal clear pathogenic genotypes also in large pedigrees. To the best of our knowledge, this is the first genetic study of LQTS patients from Russian families.

In vitro biosafety profile evaluation of multipotent mesenchymal stem cells derived from the bone marrow of sarcoma patients.

BACKGROUND: In osteosarcoma (OS) and most Ewing sarcoma (EWS) patients, the primary tumor originates in the bone. Although tumor resection surgery is commonly used to treat these diseases, it frequently leaves massive bone defects that are particularly difficult to be treated. Due to the therapeutic potential of mesenchymal stem cells (MSCs), OS and EWS patients could benefit from an autologous MSCs-based bone reconstruction. However, safety concerns regarding the in vitro expansion of bone marrow-derived MSCs have been raised. To investigate the possible oncogenic potential of MSCs from OS or EWS patients (MSC-SAR) after expansion, this study focused on a biosafety assessment of MSC-SAR obtained after short- and long-term cultivation compared with MSCs from healthy donors (MSC-CTRL). METHODS: We initially characterized the morphology, immunophenotype, and differentiation multipotency of isolated MSC-SAR. MSC-SAR and MSC-CTRL were subsequently expanded under identical culture conditions. Cells at the early (P3/P4) and late (P10) passages were collected for the in vitro analyses including: sequencing of genes frequently mutated in OS and EWS, evaluation of telomerase activity, assessment of the gene expression profile and activity of major cancer pathways, cytogenetic analysis on synchronous MSCs, and molecular karyotyping using a comparative genomic hybridization (CGH) array. RESULTS: MSC-SAR displayed comparable morphology, immunophenotype, proliferation rate, differentiation potential, and telomerase activity to MSC-CTRL. Both cell types displayed signs of senescence in the late stages of culture with no relevant changes in cancer gene expression. However, cytogenetic analysis detected chromosomal anomalies in the early and late stages of MSC-SAR and MSC-CTRL after culture. CONCLUSIONS: Our results demonstrated that the in vitro expansion of MSCs does not influence or favor malignant transformation since MSC-SAR were not more prone than MSC-CTRL to deleterious changes during culture. However, the presence of chromosomal aberrations supports rigorous phenotypic, functional and genetic evaluation of the biosafety of MSCs, which is important for clinical applications.

Validation of a quantitative PCR-high-resolution melting protocol for simultaneous screening of COL1A1 and COL1A2 point mutations and large rearrangements: application for diagnosis of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a connective tissue disorder mostly characterized by autosomal dominant inheritance. Over 1,100 causal mutations have been identified scattered along all exons of genes encoding type I collagen precursors, COL1A1 and COL1A2. Because of the absence of mutational hotspots, Sanger sequencing is considered the gold standard for molecular analysis even if the workload is very laborious and expensive. To overcome this issue, different prescreening methods have been proposed, including DHPLC and biochemical studies on cultured dermal fibroblasts; however, both approaches present different drawbacks. Moreover, in case of patients who screen negative for point mutations, an additional screening step for complex rearrangements is required; the added causative variants expected from this approach are about 1-2%. The aim of this study was to optimize and validate a new protocol that combines quantitative PCR (qPCR) and high-resolution melting (HRM) curve analysis to reduce time and costs for molecular diagnosis. Results of qPCR-HRM screening on 57 OI patients, validated by DHPLC-direct sequencing and multiplex ligation-dependent probe amplification (MLPA), indicate that all alterations identified with the mentioned methodologies are successfully detected by qPCR-HRM. Moreover, HRM was able to discriminate complex genotypes and homozygous variants. Finally, qPCR-HRM outperformed direct sequencing and DHPLC-MLPA in terms of rapidity and costs.

Biological indicators of prognosis in Ewing's sarcoma: an emerging role for lectin galactoside-binding soluble 3 binding protein (LGALS3BP).

Starting from an experimental model that accounts for the 2 most important adverse processes to successful therapy of Ewing's sarcoma (EWS), chemoresistance and the presence of metastasis at the time of diagnosis, we defined a molecular signature of potential prognostic value. Functional annotation of differentially regulated genes revealed 3 major networks related to cell cycle, cell-to-cell interactions and cellular development. The prognostic impact of 8 genes, representative of these 3 networks, was validated in 56 EWS patients. High mRNA expression levels of HINT1, IFITM2, LGALS3BP, STOML2 and c-MYC were associated with reduced risk to death and lower risk to develop metastasis. At multivariate analysis, LGALS3BP, a matricellular protein with a role in tumor progression and metastasis, was the most important predictor of event-free survival and overall survival. The association between LGALS3BP and prognosis was confirmed at protein level, when expression of the molecule was determined in tumor tissues but not in serum, indicating a role for the protein at local tumor microenvironment. Engineered enhancement of LGALS3BP expression in EWS cells resulted in inhibition of anchorage independent cell growth and reduction of cell migration and metastasis. Silencing of LGALS3BP expression reverted cell behavior with respect to in vitro parameters, thus providing further functional validation of genetic data obtained in clinical samples. Thus, we propose LGALS3BP as a novel reliable indicator of prognosis, and we offer genetic signatures to the scientific communities for cross-validation and meta-analysis, which are indispensable tools for a rare tumor such as EWS.

Multiple osteochondromas: mutation update and description of the multiple osteochondromas mutation database (MOdb).

Multiple osteochondromas (MO) is an autosomal dominant skeletal disease characterized by the formation of multiple cartilage-capped bone tumors growing outward from the metaphyses of long tubular bones. MO is genetically heterogeneous, and is associated with mutations in Exostosin-1 (EXT1) or Exostosin-2 (EXT2), both tumor-suppressor genes of the EXT gene family. All members of this multigene family encode glycosyltransferases involved in the adhesion and/or polymerization of heparin sulfate (HS) chains at HS proteoglycans (HSPGs). HSPGs have been shown to play a role in the diffusion of Ihh, thereby regulating chondrocyte proliferation and differentiation. EXT1 is located at 8q24.11-q24.13, and comprises 11 exons, whereas the 16 exon EXT2 is located at 11p12-p11. To date, an EXT1 or EXT2 mutation is detected in 70-95% of affected individuals. EXT1 mutations are detected in +/-65% of cases, versus +/-35% EXT2 mutations in MO patient cohorts. Inactivating mutations (nonsense, frame shift, and splice-site mutations) represent the majority of MO causing mutations (75-80%). In this article, the clinical aspects and molecular genetics of EXT1 and EXT2 are reviewed together with 895 variants in MO patients. An overview of the reported variants is provided by the online Multiple Osteochondromas Mutation Database (http://medgen.ua.ac.be/LOVD).

Prognostic value of CCN3 in osteosarcoma.

PURPOSE: Osteosarcoma, the most common bone tumor, lacks prognostic markers that could distinguish patients before therapy and drive treatment choices. We assessed the prognostic value of CCN1, CCN2, and CCN3 genes, involved in fundamental biological processes. EXPERIMENTAL DESIGN: Expression of CCN1, CCN2, and CCN3 was measured by quantitative PCR in 45 newly diagnosed osteosarcomas. Cancer-specific survival was estimated using the Kaplan-Meier method. Associations with osteoblastic differentiation and/or drug response genes were assessed in tumor cells using Spearman correlation and Fisher's exact tests. RESULTS: CCN1 and CCN2 expression was associated with genes involved in commitment of mesenchymal stem cells toward osteoblasts and in early phases of osteoblastic differentiation (RUNX family genes; cadherin 4, 11, and 13; jun and fos; collagen I and SPARC). Although CCN3 is barely expressed in normal proliferating osteoblasts and mesenchymal stem cells, its expression was generally high in osteosarcoma and its level of expression did not correlate with any specific osteoblastic differentiation genes. High expression of CCN3 significantly correlated with worse prognosis in osteosarcoma. This may be only partly explained by the association with the expression of multidrug resistance-related protein 1 and 4, two ATP-binding cassette transporters that also acted as predictors of worse outcome in our study. CONCLUSIONS: Our study showed temporal and coordinated expression of CCN1, CCN2, and CCN3 genes during osteoblastic differentiation and highlighted significant differences between human normal and osteosarcoma cell differentiation in vitro. CCN1 and CCN2 expression shows no prognostic relevance in osteosarcoma. In contrast, assessment for CCN3 expression levels at diagnosis may represent a useful molecular tool to early identification of patients with different prognosis.

CD99 acts as an oncosuppressor in osteosarcoma.

CD99 was recently reported to be under control of the osteoblast-specific transcription factor Cbfa1 (RUNX2) in osteoblasts, suggesting a role in the phato-physiology of these cells. No extensive information is available on the role(s) of this molecule in malignant phenotype, and osteosarcoma, in particular, has never been studied. We report that in 11 different cell lines and 17 clinical samples CD99 expression is either undetectable or very low. Being expressed in the normal counterpart, we tested the hypothesis that CD99 down-regulation may have a role in osteosarcoma development and progression. CD99-forced expression in two osteosarcoma cell lines significantly reduced resistance to anoikis, inhibited growth in anchorage independence as well as cell migration, and led to abrogation of tumorigenic and metastatic ability. Therefore, the molecule acts as a potent suppressor of malignancy in osteosarcoma. CD99 gene transfection induces caveolin-1 up-regulation and the two molecules were found to colocalize on the cell surface. Treatment with antisense oligonucleotides to caveolin-1 abrogates the effects of CD99 on migration. The findings point to an antioncogenic role for CD99 in osteosarcoma, likely through the regulation of caveolin-1 and inhibition of c-Src kinase activity.

Caveolin-1 reduces osteosarcoma metastases by inhibiting c-Src activity and met signaling.

Caveolin-1 (Cav-1) is highly expressed in normal osteoblasts. This article reports that Cav-1 down-regulation is part of osteoblast transformation and osteosarcoma progression and validates its role as oncosuppressor in human osteosarcoma. A survey of 6-year follow-up indicates a better overall survival for osteosarcoma expressing a level of Cav-1 similar to osteoblasts. However, the majority of primary osteosarcoma shows significantly lower levels of Cav-1 than normal osteoblasts. Accordingly, Met-induced osteoblast transformation is associated with Cav-1 down-regulation. In vitro, osteosarcoma cell lines forced to overexpress Cav-1 show reduced malignancy with inhibited anchorage-independent growth, migration, and invasion. In vivo, Cav-1 overexpression abrogates the metastatic ability of osteosarcoma cells. c-Src and c-Met tyrosine kinases, which are activated in osteosarcoma, colocalize with Cav-1 and are inhibited on Cav-1 overexpression. Thus, Cav-1 behaves as an oncosuppressor in osteosarcoma. Altogether, data suggest that Cav-1 down-modulation might function as a permissive mechanism, which, by unleashing c-Src and Met signaling, enables osteosarcoma cells to invade neighboring tissues. These data strengthen the rationale to target c-Src family kinases and/or Met receptor to improve the extremely poor prognosis of metastatic osteosarcoma.

Bilateral Symmetry of Visual Function Loss in Cone-Rod Dystrophies.

PURPOSE: To investigate bilateral symmetry of visual impairment in cone-rod dystrophy (CRD) patients and understand the feasibility of clinical trial designs treating one eye and using the untreated eye as an internal control. METHODS: This was a retrospective study of visual function loss measures in 436 CRD patients followed at the Ophthalmology Department of the Catholic University in Rome. Clinical measures considered were best-corrected visual acuity, focal macular cone electroretinogram (fERG), and Ganzfeld cone-mediated and rod-mediated electroretinograms. Interocular agreement in each of these clinical indexes was assessed by t- and Wilcoxon tests for paired samples, structural (Deming) regression analysis, and intraclass correlation. Baseline and follow-up measures were analyzed. A separate analysis was performed on the subset of 61 CRD patients carrying likely disease-causing mutations in the ABCA4 gene. RESULTS: Statistical tests show a very high degree of bilateral symmetry in the extent and progression of visual impairment in the fellow eyes of CRD patients. CONCLUSIONS: These data contribute to a better understanding of CRDs and support the feasibility of clinical trial designs involving unilateral eye treatment with the use of fellow eye as internal control.

Inhibition of angiogenic activity of renal carcinoma by an antisense oligonucleotide targeting fibroblast growth factor-2.

BACKGROUND: Fibroblast growth factor-2 (FGF-2) induces angiogenesis, critical for the growth and metastatic spread of tumors. MATERIALS AND METHODS: The effect of blocking FGF-2 synthesis by an antisense phosphorothioate oligodeoxynucleotide (PS-ODN2) was evaluated on the angiogenic activity of Caki-1 and of a cell line isolated from a renal carcinoma bone metastasis (CRBM-1990). After the transfection with PS-ODN2, FGF-2 mRNA, protein expression and angiogenic activity were evaluated. RESULTS: In Caki-1, a not significant decrease in the released FGF-2 was observed after 72 hours. In CRBM-1990, a not significant decrease in intracellular FGF-2 protein was observed after 72 hours. Endothelial cell migration induced by the conditioned media from Caki-1 treated with PS-ODN2 for 72 hours was significantly reduced. CONCLUSION: PS-ODN2 treatment of the established line Caki-1 induced minimal variations in FGF-2 expression, but inhibited endothelial cell migration. In CRBM-1990 cells, PS-ODN2 determined a decrease in intracellular protein without reducing the ability to induce endothelial cell migration and proliferation.

Identification and functional characterization of the human EXT1 promoter region.

BACKGROUND: Mutations in Exostosin-1 (EXT1) or Exostosin-2 (EXT2) cause the autosomal dominant disorder multiple osteochondromas (MO). This disease is mainly characterized by the appearance of multiple cartilage-capped protuberances arising from children's metaphyses and is known to display clinical inter- and intrafamilial variations. EXT1 and EXT2 are both tumor suppressor genes encoding proteins that function as glycosyltransferases, catalyzing the biosynthesis of heparan sulfate. At present, however, very little is known about the regulation of these genes. Two of the most intriguing questions concerning the pathogenesis of MO are how disruption of a ubiquitously expressed gene causes this cartilage-specific disease and how the clinical intrafamilial variation can be explained. Since mutations in the EXT1 gene are responsible for ~65% of the MO families with known causal mutation, our aim was to isolate and characterize the EXT1 promoter region to elucidate the transcriptional regulation of this tumor suppressor gene. METHODS: In the present study, luciferase reporter gene assays were used to experimentally confirm the in silico predicted EXT1 core promoter region. Subsequently, we evaluated the effect of single nucleotide polymorphisms (SNP's) on EXT1 promoter activity and transcription factor binding using luciferase assays, electrophoretic mobility shift assays (EMSA), and enzyme-linked immunosorbent assays (ELISA). Finally, a genotype-phenotype study was performed with the aim to identify one or more genetic modifiers influencing the clinical expression of MO. RESULTS: Transient transfection of HEK293 cells with a series of luciferase reporter constructs mapped the EXT1 core promoter at approximately -917 bp upstream of the EXT1 start codon, within a 123 bp region. This region is conserved in mammals and located within a CpG-island containing a CAAT- and a GT-box. A polymorphic G/C-SNP at -1158 bp (rs34016643) was demonstrated to be located in a USF1 transcription factor binding site, which is lost with the presence of the C-allele resulting in a ~56% increase in EXT1 promoter activity. A genotype-phenotype study was suggestive for association of the C-allele with shorter stature, but also with a smaller number of osteochondromas. CONCLUSIONS: We provide for the first time insight into the molecular regulation of EXT1. Although a larger patient population will be necessary for statistical significance, our data suggest the polymorphism rs34016643, in close proximity of the EXT1 promoter, to be a potential regulatory SNP, which could be a primary modifier that might explain part of the clinical variation observed in MO patients.

Genotype-phenotype correlation study in 529 patients with multiple hereditary exostoses: identification of "protective" and "risk" factors.

BACKGROUND: Multiple hereditary exostoses is an autosomal dominant skeletal disorder characterized by wide variation in clinical phenotype. The aim of this study was to evaluate whether the severity of the disease is linked with a specific genetic background. METHODS: Five hundred and twenty-nine patients with multiple hereditary exostoses from two different European referral centers participated in the study. According to a new clinical classification based on the presence or absence of deformities and functional limitations, the phenotype of the patients was assessed as mild (the absence of both aspects), intermediate, or severe (the concurrent presence of both aspects). An identical molecular screening protocol with denaturing high-performance liquid chromatography and multiplex ligation-dependent probe amplification was performed in both institutions. RESULTS: In our cohort of patients, variables such as female sex (odds ratio = 1.840; 95% confidence interval, 1.223 to 2.766), fewer than five skeletal sites with exostoses (odds ratio = 7.588; 95% confidence interval, 3.479 to 16.553), EXT2 mutations (odds ratio = 2.652; 95% confidence interval, 1.665 to 4.223), and absence of EXT1/2 mutations (odds ratio = 1.975; 95% confidence interval, 1.051 to 3.713) described patients with a mild phenotype; in contrast, a severe phenotype was associated with male sex (odds ratio = 2.431; 95% confidence interval, 1.544 to 3.826), EXT1 mutations (odds ratio = 6.817; 95% confidence interval, 1.003 to 46.348), and more than twenty affected skeletal sites (odds ratio = 2.413; 95% confidence interval, 1.144 to 5.091). Malignant transformation was observed in 5% of patients, and no evidence of association between chondrosarcoma onset and EXT mutation, sex, severity of disease, or number of lesions was detected. CONCLUSIONS: The identified "protective" and "risk" factors, as well as the proposed classification system, represent helpful tools for clinical management and follow-up of patients with multiple hereditary exostoses; moreover, homogeneous cohorts of patients, useful for studies on the pathogenesis of multiple hereditary exostoses, have been identified.

Overcoming glutathione S-transferase P1-related cisplatin resistance in osteosarcoma.

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most used drugs for osteosarcoma chemotherapy. By using a series of CDDP-resistant variants, which were established from the U-2OS and Saos-2 human osteosarcoma cell lines, we found that CDDP resistance was mainly associated with the increase of both the intracellular level and enzymatic activity of glutathione S-transferase P1 (GSTP1). On the basis of these findings, we evaluated the clinical effect of GSTP1 in a series of 34 high-grade osteosarcoma patients and we found that the increased expression of GSTP1 gene was associated with a significantly higher relapse rate and a worse clinical outcome. These indications prompted us to assess the in vitro effectiveness of 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX), a promising new anticancer agent that is a highly efficient inhibitor of GSTP1. NBDHEX was tested on a panel of 10 human osteosarcoma cell lines and 20 variants of the U-2OS or Saos-2 cell lines that were resistant to CDDP, doxorubicin, or methotrexate. NBDHEX proved to be very active on the vast majority of these cell lines, including those with higher GSTP1 levels and enzymatic activity. Drug combination studies showed that NBDHEX can be used in association with CDDP and provided useful information about the best modality of their combined administration. In conclusion, our findings show that GSTP1 has a relevant effect for both CDDP resistance and clinical outcome of high-grade osteosarcoma and that targeting GSTP1 with NBDHEX may be considered a promising new therapeutic possibility for osteosarcoma patients who fail to respond to conventional chemotherapy.

Insulin-like growth factor binding protein 3 as an anticancer molecule in Ewing's sarcoma.

The IGF/IGF-IR system plays a major role in the pathogenesis and progression of Ewing's sarcoma. In this article, the authors evaluated whether the insulin-like growth factor binding protein-3 (IGFBP-3), a molecule with growth-inhibitory and proapoptotic activities, may be exploited for therapeutic applications in the treatment of Ewing's sarcoma (EWS). Expression of IGFBP-3 was analyzed in a panel of EWS cell lines and clinical samples. Parameters related to malignancy (growth in anchorage-independent conditions, migration, invasion and angiogenesis properties) were studied to establish the potential in vitro anticancer effects of exogenous IGFBP-3. The activity of the molecule against metastasis was analyzed by taking advantage of selected clones in which IGFBP-3 endogenous production was induced by gene transfection. The authors observed a generally low expression of IGFBP-3 either in a panel of EWS cell lines or clinical samples. Exogenous IGFBP-3 remarkably inhibits EWS growth, both in monolayer and anchorage-independent conditions, and significantly reduces cell motility. Forced expression of IGFBP-3 in TC-71 EWS cells confirms the growth and migratory effects observed with the exogenous protein, decreases the production or activity of the matrixmetalloprotease MMP-9 and vascular endothelial factor (VEGF)-A and abrogates EWS metastasis ability. IGFBP-3 acts mainly through IGF-dependent mechanisms and the protein may therefore represent an alternative strategy to inhibit IGF-IR functions. The data indicate IGFBP-3 as a molecule of therapeutic potential in EWS.