Genetic predisposition to pleural mesothelioma. Research on genetic risk factors in the population of Casale Monferrato

SUMMARY OF THE RESEARCH PROJECT
FUNDED BY THE BUZZI UNICEM FOUNDATION

PROJECT LEADER:
Prof. Irma Dianzani

SITE:
University of Novara

PROJECT STATUS:
Completed

RESEARCH SUMMARY:
BACKGROUND AND RATIONAL 
Malignant pleural mesothelioma (MM) is a rare, very aggressive tumor associated with exposure to asbestos.  Only 10% of the people exposed to high levels of asbestos develop MM.  This behavior, together with the tendency for it to run in families, suggests that there is a genetic predisposition, but the rarity of the tumor has prevented large-scale genetic studies from being conducted thus far (Neri et al 2008; Ugolini et al. 2008).  Asbestos fibers are thought to be cancerogenic through different mechanisms: 1) mechanical effects, such as interference with the formation of the mitotic spindle and chromosome segregation; 2) generation of oxygen reactive species either from the surface of the fibers after reactions involving iron as a catalyst or as a result of frustrated phagocytosis; 3) localized activation of intracellular signaling pathways (promoting effect).  Consequences from the oxidative damage include DNA strand breakage and base modifications (Jensen, et al 1996; Kamp et al 1999; Wang et al 1998; Yang et al. 2006).
For several years, we have been evaluating the role of genetic susceptibility in the development of MM.  We designed an association study of single nucleotide polymorphisms (SNPs) in individuals with MM and controls.  
The first cohort consisted of people from Casale Monferrato, a city with a high environmental risk for the presence of asbestos from the cement industry until 1986 (Magnani et al. 2001). This was a case-control study, in which the patients and controls were all residing in Casale Monferrato and the surrounding areas.  Since the city has never been subject to large-scale immigration, the population is considered to be relatively homogeneous. Over the course of the years, study has enrolled 151 patients and 252 controls thus far.  
The controls were recruited from National Health System registries.  Both the patients and the controls were interviewed by an expert epidemiologist and their exposure to asbestos was thoroughly evaluated by an industrial hygienist.  A similar study was also initiated in Turin in collaboration with Dr. Dario Mirabelli and has thus far enrolled 100 patients and 60 controls. The controls in this case were selected from patients who had been admitted to hospital for non-cancerous reasons. 
To further increase the number of patients and controls, we also initiated a collaboration with Dr. Stefano Bonassi’s group in Roma and Dr. Donatella Ugolini from ISTGE, who provided a cohort of 100 patients and 100 controls from Genoa. 
Lastly, we initiated a collaboration with Dr. Hirvonen who provided 100 patients and 100 controls from Finland.
We enrolled a total of 451 patients and 512 controls. We did a thorough evaluation of the exposure to asbestos in each of the four cohorts. 
The purpose of the study was to analyze the SNPs in genes that could play a role in the carcinogenic activity of asbestos (genes involved in DNA repair, in controlling the redox state of the cell, in inflammation.  The SNPs were selected based on the following criteria: a) some polymorphisms had  already been associated with functional alterations in other studies; b) the SNPs led to non-conservative replacements localized in important functional domains of protein or suggestive of alteration in the splicing or transcription mechanism. 
The composite genotype of each individual was individually evaluated. 
A preliminary study conducted in 81 patients and 110 controls from Casale (funded by the Piedmont Region) showed an association between XRCC1 399Q and MM (Dianzani et al. 2006).  We subsequently confirmed this association in a larger cohort from Casale Monferrato (133 patients, 182 controls; increase in the mutated alleles: OR=1.46; 95%IC 1.01-2.12) (Betti et al. under review).  In another study, we investigated the association between MM and the NAT2 gene, which encodes the N-acetyl-transferase 2 enzyme. A Finish study observed an association with the slow acetylator phenotype, while an Italian study observed an association with the fast acetylator phenotype.  We studied a cohort of 252 patients and 262 controls recruited from Casale Monferrato and Liguria.  The fast acetylator phenotype demonstrated an increased OR, although it was not statistically significant in either the subjects exposed to asbestos (OR=1.47; 95%IC 0.96–2.26) or the general population (OR=1.38; 95%IC 0.93–2.04). This data shows that this phenotype does not play an important role in the pathogenesis of MM (Betti et al. 2009).

OBJECTIVES AND RESULTS 
The first part of the study was to evaluate the results previously obtained in a larger cohort and extend the number of SNPs under investigation.  We studied 35 SNPs localized in 15 genes that could be involved in the pathogenesis of mesothelioma.  The genes under investigation are involved in: DNA repair (XRCC1, XRCC3, ERCC1, ERCC2, OGG1, PARP, PCNA, MGMT, APEX, NBS1), controlling the redox state (GPX1, SOD2), inflammation (OPN), a phosphatase 1 gene (RAI) and SEP15, a gene that is down-regulated in mesothelioma cells.  Four of the above genes had already been previously studied (Dianzani et al. 2006).
The SNPs were analyzed in the cohort from Casale Monferrato (151 patients and 252 controls) and in a prior cohort from Turin (69 patients and 44 controls). 
The current results confirm that XRCC1 399Q and XRCC1 -77T may play a role as risk factors for MM.  In the subjects exposed to asbestos, the increase in the Q alleles demonstrate an OR=1.44; 95%IC 1.02–2.03 and the increase in the T alleles (from heterozygous to homozygous) show an OR=1.3; 95% IC 0.98–1.8.  The haplotype, which includes these two SNPs, was also associated with MM (OR=1.76; 95% IC 1.04–2.96). 
Since we know that the two above SNPs can modify the three-dimensional structure and the quantitative expression of the XRCC1 protein, we can hypothesize that the association with MM is caused by a pathogenic mechanism. 
An association was also found for the ERCC1 gene, which is located in a region adjacent to XRCC1 on the 19q13.2 chromosome. Both the XRCC1 and ERCC1 genes are involved in DNA repair. 
None of the other SNPs demonstrated a statistically significant OR. 
These study results were submitted to the Mutation Research journal, which requested a few small modifications and is currently under review (Betti et al. under review).
The purpose of the second part of the project funded by the AIRC, the Piedmont Region and the Buzzi Foundation was to systematically identify all the risk factors for developing MM (Genome Wide Association Study-GWAS) using microarrays from Illumina which can analyze the entire genome.  This strategy has been used in many studies to identify risk factors for other tumor types (Rothman et al. 2010), so we initiated a collaboration with Prof. Giuseppe Matullo from Turin who has Illumina equipment for analyzing SNPs.  We analyzed the cohort from Casale, Turin and Genoa (367 patients and 392 controls) using a microarray that can interrogate 370,000 genomic variations and CNVs (copy number variations). This study led to the identification of 20 SNPs with OR between 2 and 5 and p≤0.00005.  These results should be confirmed in an independent cohort.

OTHER FUNDING OBTAINED FOR THIS PROJECT 
For the study on the candidate genes: Regione Piemonte Ricerca Sanitaria Finalizzata 2006, 2007, 2008 (all to I.Dianzani); for the GWAS: Regione Piemonte Ricerca Sanitaria Finalizzata 2009 (to I.Dianzani), AIRC 2007 (to S.Bonassi).

TRANSLATIONAL IMPLICATIONS OF THE PROJECT 
identifying the genes and variations involved will allow us to better determine the risk of developing MM from exposure to asbestos and better clarify the pathogenetic bases. 

PUBLICATIONS:
Dianzani I., Gibello L., Biava A., Giordano M., Bertolotti M., Betti M., Ferrante D., Guarrera S., Betta G.P., Mirabelli D., Matullo G., Magnani C., 2006. Polymorphisms in DNA repair genes as risk factors for asbestos-related malignant mesothelioma in a general population study. Mutat. Res. 599, 124-134.

Neri M., Ugolini D., Dianzani I., Filiberti R., Gemignani F., Landi S., Magnani C., Mutti L., Puntoni R., Bonassi S. 2008 Genetic susceptibility to malignant pleural mesothelioma and other asbestos-related diseases. Mutat. Res. 659, 126-136.

Ugolini D, Neri M, Ceppi M, Cesario A, Dianzani I, Filiberti R, Gemignani F, Landi S, Magnani C, Mutti L, Puntoni R, Bonassi S., 2008 Genetic susceptibility to malignant mesothelioma and exposure to asbestos: The influence of the familial factor. Mutation Research 658, 162–171.

Betti M., Neri M., Ferrante D., S. Landi, Biava A., Gemignani F., Bertolotti M., Mirabelli D., Padoan M.,Ugolini D., Bonassi S., Magnani C., Dianzani I. 2009 Pooled analysis of NAT2 genotypes as risk factors for asbestos-related malignant mesothelioma. Int. J. Hyg. Environ. Health 212, 322-329.

Betti M., Ferrante D., Padoan M., Guarrera S., Giordano M., Aspesi A., Mirabelli D., Casadio C., Ardissone F., Ruffini E., Betta P.G., Libener R., Guaschino R., Matullo G., Piccolini E., Magnani C., Dianzani I. XRCC1 and ERCC1 variants modify malignant mesothelioma risk: A case-control study inviato a Mutation Research in corso di revisione.