Mauro Tognon

Strong Evidence of Linkage Disequilibrium between Polymorphisms at the IRF6 Locus and Nonsyndromic Cleft Lip With or Without Cleft Palate, in an Italian Population

Cleft lip with or without cleft palate (CL/P) is one of the most common birth defects, but its etiology is largely unknown. It is very likely that both genetic and environmental factors contribute to this malformation. Mutations in the gene for interferon regulatory factor 6 (IRF6) have been shown to be the cause of Van der Woude syndrome, a dominant disorder that has CL/P as a common feature. Recently, it has been reported that genetic polymorphisms at the IRF6 locus are associated with nonsyndromic CL/P, with stronger association in Asian and South American populations. We investigated four markers spanning the IRF6 locus, using the transmission/disequilibrium test. A sample of 219 Italian triads of patients and their parents were enrolled in the study. Strong evidence of linkage disequilibrium was found between markers and disease in both single-allele (P=.002 at marker rs2235375) and haplotype (P=.0005) analyses. These findings confirm the contribution of IRF6 in the etiology of nonsyndromic CL/P and strongly support its involvement in populations of European ancestry.

SV40 replication in human mesothelial cells induces HGF/Met receptor activation: A model for viral-related carcinogenesis of human malignant mesothelioma

Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation.

High Levels of Antibodies Against Merkel Cell Polyomavirus Identify a Subset of Patients With Merkel Cell Carcinoma With Better Clinical Outcome

PURPOSE A new human polyomavirus, Merkel cell polyomavirus (MCV), was identified in 2008 in tumor tissue of patients with Merkel cell carcinoma (MCC), a relatively rare human skin cancer. In this study, we investigated patients with MCC and controls for the presence of antibodies against MCV and their association with clinical characteristics. PATIENTS AND METHODS Antibodies against MCV were investigated by enzyme-linked immunosorbent assay in 68 patients with MCC and 82 controls using VP1 virus-like particles produced in insect cells. RESULTS Antibodies against MCV were detected in all patients with MCC and in 85% of controls. However, high antibody titers (> 10,000) were rarely observed in controls (7.3%) and they were detected in 64.7% of patients with MCC (P < .001) in contrast to the absence of VP1 expression in tumor samples. In addition, the geometric mean titer of anti-MCV in patients with MCC was around 14 times higher than that observed in MCV-positive controls (P < .001) and was not correlated with tumor viral load. High antibody titers were not found to be associated with any subject or tumor characteristics, but better progression-free survival was observed in patients with high antibody titers (hazard ratio, 4.6; 95% CI, 1.7 to 12.2; P = .002). CONCLUSION High titers of MCV antibodies in a much higher proportion of patients with MCC than in controls confirmed the association between MCV infection and MCC. The findings also indicated that a better progression-free survival occurred in patients with high MCV antibody titers and suggested that there are at least two distinct etiologic causes of MCC.

BK and JC Human Polyomaviruses and Simian Virus 40: Natural History of Infection in Humans, Experimental Oncogenicity, and Association with Human Tumors

Publisher Summary Several viruses are ubiquitous in the human population and produce a persistent or latent infection in humans. Some of them—such as human polyomaviruses BK (BKV) and JC (JCV)—are oncogenic in experimental systems and are associated with human tumors. This chapter considers the general properties of the human polyomaviruses BK and JC, the characteristics of the latent infection and of the ubiquitous state of these viruses in humans, their transforming capacity in vitro , their oncogenicity in experimental animals, and their possible etiologic role in human tumors are also evaluated. It is noted that the classic Kochs postulates cannot be applied to latent viruses. New rules are considered for these viruses to establish their oncogenic role. It is reported that BKV fulfills these criteria, suggesting that BKV may cooperate, under peculiar conditions, as a cofactor in the development or progression of human tumors. BKV oncogenicity is revealed by cooperating events, such as oncogene activation, loss of tumor suppressor genes, or rearrangements of the viral genome, inducing proliferation of clonal neoplastic cells in a population of latently BKV-infected cells.

Oncogenic transformation by BK virus and association with human tumors.

BK virus (BKV), a human polyomavirus closely related to JC virus and Simian Virus 40, is ubiquitous in human populations worldwide. After primary infection, BKV establishes a lifelong latent infection in many organs. BKV transforms rodent cells to the neoplastic phenotype and is highly oncogenic in rodents. This review considers the oncogenic potential of BKV in humans and its possible involvement in human tumors. BKV sequences and T antigen (Tag) are detected in several types of human neoplasms, although the viral load is generally low, with less than one copy of the viral genome per cell. The possible causative role of BKV in human oncogenesis rests on the ability of BKV Tag to inactivate the functions of tumor suppressor proteins p53 and pRB family as well as on its ability to induce chromosomal aberrations in human cells. A ‘hit and run’ mechanism and secretion of paracrine growth factors by BKV Tag-positive cells, recruiting into proliferation neighboring and distant cells, are discussed as possible BKV pathogenic elements in human oncogenesis.

C677T variant form at the MTHFR gene and CL/P: A risk factor for mothers?

Maternal folic acid supplementation in early pregnancy has been suggested to play a role in the prevention of nonsyndromic orofacial cleft, i.e., cleft lip with or without cleft palate (CL/P). Moreover, some authors demonstrated association of the C-->T mutation (C677T), converting an alanine to a valine residue in 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, with other congenital anomalies such as neural tube defects (NTDs). Because of MTHFRs involvement in the metabolism of folate, we investigated 64 CL/P patients and their parents for C677T MTHFR mutation. No linkage disequilibrium was found using the transmission disequilibrium test (TDT). However, a significantly higher mutation frequency was detected in mothers of CL/P patients compared to controls. The odds ratios calculated for mothers having CT or TT genotype, compared to the normal CC genotype, were 2.75 (95% confidence interval 1.30-5.57) and 2.51 (1.00-6.14), respectively. These results support the involvement of the folate pathway in the etiology of CL/P, and indicate an effect of the maternal genotype, rather than influence of the embryos genotype.

Age-Specific Seroprevalences of Merkel Cell Polyomavirus, Human Polyomaviruses 6, 7, and 9, and Trichodysplasia Spinulosa-Associated Polyomavirus

ABSTRACT Six new human polyomaviruses have been identified since 2008 (Merkel cell polyomavirus [MCPyV], human polyomavirus 6 [HPyV6], HPyV7, HPyV9, trichodysplasia spinulosa polyomavirus [TSPyV], and Malawi polyomavirus [MWPyV]). The presence of specific antibodies against MCPyV, HPyV6, HPyV7, HPyV9, and TSPyV in 828 Italian subjects aged 1 to 100 years was investigated by virus-like particle-based enzyme-linked immunosorbent assays (ELISAs). The findings indicate that all of these new polyomaviruses circulate widely in humans, with seroprevalences in adulthood ranging from 39.4% for HPyV9 to 87.1% for MCPyV, and that primary exposure is most intense in childhood, with the exception of HPyV7 and HPyV9, for which the seroprevalence increased throughout life. The proportion of subjects with high antibody titers was found to increase with age for MCPyV and to decrease with age for TSPyV.

Generation of Merkel Cell Polyomavirus (MCV)-Like Particles and Their Application to Detection of MCV Antibodies

ABSTRACT The genome of a new human polyomavirus, known as Merkel cell polyomavirus (MCV), has recently been reported to be integrated within the cellular DNA of Merkel cell carcinoma (MCC), a rare human skin cancer. To investigate MCV seroprevalence in the general population, we expressed three different MCV VP1 in insect cells using recombinant baculoviruses. Viruslike particles (VLPs) were obtained with only one of the three VP1 genes. High-titer antibodies against VP1 VLPs were detected in mice immunized with MCV VLPs, and limited cross-reactivity was observed with BK polyomavirus (BKV) and lymphotropic polyomavirus (LPV). MCV antibodies were detected in 77% of the general population, with no variations according to age.

SV40 Enhances the Risk of Malignant Mesothelioma among People Exposed to Asbestos: A Molecular Epidemiologic Case-Control Study

We conducted a case-control study on asbestos exposure and presence of SV40 in tumor samples of malignant mesotheliomas (MMs) and bladder urotheliomas (BUs). PCR analysis revealed the presence of SV40 DNA (SV40+) in eight (42.1%) MMs and 6 (33.3%) BUs. The odds ratio for MM Asb- and SV40+ was 0.4 [95% confidence interval (95% CI), 0.03-4.0], for Asb+ and SV40- was 3.6 (95% CI, 0.6-21.0), and for Asb+ and SV40+ was 12.6 (95% CI, 1.2-133.9). Our results suggest that SV40 increases the risk of MM among individuals exposed to asbestos.

Recent developments in orofacial cleft genetics.

Nonsyndromic cleft of the lip and/or palate (CLP or orofacial cleft) derives from an embryopathy with consequent failure of the nasal process and/or palatal shelves fusion. This severe birth defect is one of the most common malformations among live births. Nonsyndromic CLP is composed of two separate entities: cleft lip and palate (CL±P) and cleft palate only (CPO). Both have a genetic background, and environmental factors probably disclose these malformations. In CL±P, several loci have been identified, and, in one case, a specific gene has also been found. In CPO, one gene has been identified, but many more are probably involved. Because of the complexity of the genetics of nonsyndromic CLP as a result of the difference between CL±P and CPO, heterogeneity of each group caused by the number of involved genes, type of inheritance, and interaction with environmental factors, we discuss the more sound results obtained with different approaches: epidemiological studies, animal models, human genetic studies, and in vitro studies.