Summary
The pathogenesis, progress and outcomes of most common diseases, such as cardiovascular disorders, diabetes and cancer, are subject to both genetic and environmental influences. While many environmental components have been recognized in the last century, genetic and genomic components for complex diseases are only being uncovered now. With the progressive resolution of the human genome, since the publication of its first draft in 2001, knowledge is now accumulating, which will permit scientists to resolve distinctive and shared environmental and genomic components of these diseases as well as their interactions in the near future.
Unlike the rare and severe genetic defects that cause monogenic diseases, like thalassemia, haemophilia or cystic fibrosis, the genetic factors that modulate the individual susceptibility to common diseases (cardiovascular diseases, cancers, diabetes, etc.) are functionally different forms of genes (polymorphism), which generally have a modest effect on their own, but, because of their high frequency in the population and their interactions, they can be associated with a high attributable risk. Environmental factors can reveal or facilitate the expression of such susceptibility genes (leading to a trait or phenotype). Indeed, in common diseases genetic effects can be considerably amplified in the presence of triggering factors. There is now accumulating evidence that most of the susceptibility genes for common diseases do not have only an etiological role, but rather act in response to both endogenous factors including sex and age, as well as exogenous factors, such as stress, nutrients, physical environment, working conditions, socio-economic status, infections and other diseases, therapeutic and illicit drugs, etc. A better characterisation of the interactions between environmental and genetic factors is critical to understanding the pathogenesis of multifactorial diseases, which represents a major burden of morbidity and mortality for our species.
To understand the relative roles of the genetic and environmental components in the etiopathogenesis of common diseases, it is crucial to use innovative models that are statistically powerful and flexible enough to encompass the complexity of these prevalent diseases with respect to their modulation by gene-environment interactions.
As most common diseases, including hypertension, diabetes, hypercholesterolemia and even several cancers, such as that of prostate, aggregate in families, many researchers have used the family as a model to study them. However, relatives living in the same geographic location share not only genes but also the environment, which limits the power of the model and conclusions that can be drawn from statistical analyses of the data.
Migrants offer a very useful model for the study of genes x environment interactions since they are faced with a set of novel conditions, resulting in progressive departure from original habits and the acceptance of new ways of life in their new homeland, while preserving the original genetic heritage (ancestral allelic frequency). Many studies have been performed on migrants, their diseases and outcomes in comparison to prevalence observed in their original and recipient countries. The conclusions of these studies are, however, limited mainly to the understanding of environmental impact.
We plan to combine the power of both family and migrant studies by investigating families whose members reside both in the original and in the host country in order to further dissect genetic and environmental influences.
After the Second World War, a major migration wave occurred from Southern Italy to Canada and Australia. In both cases, migrants life underwent profound changes in their new environment including differences in weather (from -25 to +25°C, mean +4°C in Montréal), in nutrition (progressive deletion of Mediterranean diet), in life style (metropolitan stress and chronobiology). Original migrants and their non-migrated siblings and cousins are at the age of cardiovascular outcomes. Differences exist between Italy and Canada in longevity, cerebrovascular and cardiac mortality, risk factors as well as in healthy years lived. However the genetic and environmental determinants of these differences are unknown at present.
In the context of this study, we will invoke a broad conception of “environment” and consider under the rubric of this term various behavioural, cognitive, affective, socio-demographic, socio-economical, nutritional and physical environmental factors relevant to cardiovascular health. We propose to study hypertension and dyslipidemia as initiating risk factors which, together with obesity, contribute to the development of CVD leading to disabling and/or fatal outcomes
On the basis of the above mentioned considerations, we are proposing the following working hypothesis:
Susceptibility and outcomes of cardiovascular and other common diseases are determined in part by environment-independent genes and for another part by the interaction of genes with factors in the environment and therefore more or less relevant in a given environment.
To test this hypothesis a trans-disciplinary team of Canadian and Italian researchers plan to study families with shared relatives residing in both Montreal and the Sannio region, in order to assess which are the main genetic and environmental factors relevant for cardiovascular determinants and outcomes.
Long term goals of these studies are:
1) to develop a model of mechanisms and outcomes of cardiovascular diseases based on the understanding of genes x environment interactions;
2) to establish new classifications of diseases resulting from resolution of gene-environment interaction patterns, thus enabling
3) better targeted management of cardiovascular diseases
4) improved public health preventive measures
Specific aims
- To identify alleles and/or haplotypes that confer same risk/protection in Sannio and Montreal;
- To identify alleles and /haplotypes that represent distinct relative risk in each geographical location;
- To identify specific environmental components that modify allelic/haplotype impacts
- To build a testable model of gene x environmental interaction.
Study population
After the Second World War, between 1950 and 1965, many migrants from Sannio, the geographical area comprising part of Campania, Molise and Puglia regions, arrived in Montreal. These new Montrealers and their descendants kept some of their home country habits, whereas they changed some others. Italian migrants in Montreal were exposed to a very different climate, lifestyle habits and, economic, social and working conditions. The typical Mediterranean diet of fruits, vegetables and olive oil was substituted at least partially by a North American diet richer in animal protein and saturated fats. In addition, most subjects came from rural areas in Italy to reside in an urban environment. Original migrants from Sannio are at an age of high prevalence for cardiovascular outcomes. This migrant population and their relatives in Sannio (non-migrants) offer a unique opportunity to examine which are the genetic determinants and the environmental determinants of cardiovascular disease, and the joint effect of genes and environmental interactions.
Study design
The AIRE files approach
The study will follow a family-based design. Eligible families will consist of relatives residing in both Sannio and Montreal with the presence of cardiovascular risk factors and/or outcomes. This design will allow us to apply linkage analysis using concordance-disconcordance equilibrium, while still preserving the power of large samples in studying contribution of target genes to the phenotype of the population. We have collected information on the migratory fluxes after second world war to date from the data files of the Anagrafe Italiani Residenti Estero(AIRE), the General Registry Office of the Italian people resident abroad founded in all communes from 1989 and periodically updated. These data allowed us to recognize the distribution of Sannio migrants in Canada and the main destination from each different Sannio village.
These data are particularly relevant in the search of eligible families, since it will allow us to target our search on the Sannio village with a consistent number of migrants arrived in Canada. The Italian Embassy in Canada and their scientific representatives are keen to collaborate in comparing the list of migrants available in the consulate office and those based on the AIRE files. This preliminary data from the AIRE confirm the feasibility of finding 150 families accomplishing the inclosing criteria within the area of interest. We have calculate that in order to enrol 150 families at least 800 families with first grade relatives living on the two side of the ocean needs to be screened.
Review of the current statistical and epidemiological data
Epidemiological and statistical data currently available on prevalence and incidence of CVD and on distribution of risk factors in Italy and in Canada, and where possible, Southern Italy and Quebec are being revised, analyzed and compared. The main sources of data are World Health Organization, Progetto Cuore from the Istituto Superiore di Sanità, Istituto Italiano di Statistica and American Hearth Association, Canadian Statistic Institute and Canadian Institute of Heart Research. Prevalence of major cardiovascular risk factors and incidence of cardiovascular disease and outcome will be compared in an analytical integrated model. Particular attention will be paid to the potential bias in assessment of prevalence and recommendations for treatment of cardiovascular risk factors in Italy, and Canada due to socio-cultural differences. In fact differences in guidelines for the detection, evaluation and treatment of arterial hypertension, dyslipidemia and diabetes must be taken into account as possible confounders in assessing prevalence of cardiovascular risk factors and therefore might have a significant impact in the differences in cardiovascular death rates between centers.
Sample size: Based on the power of the results obtained from 120 families (including 900 subjects) of French Canadians and their interfamilial differences between residence in Lac StJean-Chicoutimi region and Montréal, we plan to recruit 150 families (approximately 800 to 1000 subjects) with two living generations in both Sannio and Montreal, selected for the presence of a proband living at either site. We define a proband as:
-subject born in Sannio or with both parents born in Sannio
-diagnosed with a cardiovascular outcome
-having at least one relative with a cardiovascular outcome or risk factor
-having at least one living generation at each site (Montreal and Sannio)
-willing to participate[1].
This design will permit: a- to conduct linkage studies with affected concordant, discordant and unaffected concordant sib pairs or cousins. Extremely discordant pairs analysis has been shown to be extremely powerful for linkage studies and has already resulted produced important results for hypertension; b- while still preserving the power of large samples in studying contribution of target genes to the phenotype of the population.
Promotion program
-With the collaboration of local TV and radio a campaign for GENS promotion has being organized. Contact have been made with the RAI (Italian public national television)
- With the aid and the collaboration of local authorities meetings with the Sannio villages and cities will be organize to explain to the population the project and its aim, possibly in the context of holydays or special local events.
-Explicative and advertising materials preparation. With the aid of professionals in the field of mass communication, brochures explaining the rational and the goal of the GENS project will be prepared to be distributed throughout the Sannio area together with a first screening questionnaire.
-Implementation of the GENS website
-A toll-free number will be activated to explain the project and its goals and collect information
Promotion activities will last all along the GENS project realization, in the first phase with the aim to inform about the project and afterwards for fund raising. In the context of the present proposal the promotion activities will last for the two years.
4 – Identification of probands and family enrolment
a-to the Major office of the communes and villages of interest a letter will be mailed explaining the project and its goal and asking the permission to consult the Register office data sheets.
b-on obtainment of the major permission a team will consult the AIRE office data sheets screening for chart relative to Italian citizen migrated between after 1945 and before 1965.
It is noteworthy that the data sheets request and supply follow the current Italian laws (artt 33-34 DPR 30 maggio 1989, n. 223 “Approvazione del nuovo regolamento anagrafico della popolazione residente” e degli artt. 98, 99, 105 e 110 del Dlgs n. 196/2003 “Codice in materia di protezione dei dati personali”) which allows the release of data only and exclusively for scientific purpose. The AIRE data sheets information will be used only and exclusively within the scientific gaols of the GENS project. In addition, the data will not be released outside the scientific institution involved and the information will be rigorously denominized .
The families will be reconstructed on the base of the register office personal data sheets. Once identified an eligible data sheet holder at the Common of origin the family composition will be inferred, as well as the identification of the relatives still living in Italy. Families will be contacted directly or through local autorithies or preminent members of the communities .
-Family doctors are a very important resource for the GENS project and they will play a key role in the promotion of the project and in the search and the enrolment of the families. In fact, in a rural area of mainly small villages, family doctors as Parishes are a landmark and their collaboration will be expecially helpful in easying the contact with the population and in overcoming some natural distrust. Contact are ongoing with family doctors co-operatives in order to organize meeting to explain the project. Family doctors willing to participate could be involved in the phenotyping protocol with the support of GENS team and mobile unit. Informative material and first screening questionnaire for the identification of the probands will be supplied.
-Parish are often the historical memory of a given community so their contribution to reconstruct family history and to indentifiy probands could be critical.
The Italian Embassy in Canada and their scientific representatives are keen to collaborate in comparing the list of migrants available in the consulate office and those based on the AIRE files. In the same direction the representatives of the Sannio communities in Canada are available to compare the data from Sannio and those from the consulate offices with those of local Associations.
In Montreal, eligible probands will be identified from hospital charts including discharge codes of cardiovascular outcomes at Santa Cabrini hospital. Furthermore, with the help of the Italian community, we are planning an information campaign using Italian speaking media aimed at community acceptance and recruitment into the project.
Building the GENS network is a pre-requisite but also a product of the GENS program. In that, to proceed in the study and to recruit the families of interest to the study, largely depends on the collaboration of local and national authorities and on the sensitization of the Sannio community both in Italy and in Canada. Probands willing to participate will be asked to provide contact information about their relatives in Sannio and first and second degree relatives in Montreal and vice-versa. All contact information will be shared between Sannio and Montréal, a family will be eligible only when both the Italian and the Canadian branch will be verified and will state to be willing to participate.
It is noteworthy to point out that the strategy for searching and enrolling probands and their families will be different in Italy and in Canada due to important cultural difference, ranging from legal, ethical and social rules, life style socio-economical context. These aspects are a key part of the GENS program and comparative studies on legal aspects of population genetics and genetic studies in Italy and in Canada as well as those comparing the Italian and the Canadian health system are currently under investigation in collaboration with Prof. Biagio Grasso University of Naples “Federico II”. Dr. Giovanni Villone of the research unit of the University of Molise led by Professor Nicola Ferrara will be in charge of the ethical issues
-The organization of dedicated data base as the managment of the data and, more generally, the bioinformatics aspect of the GENS Program have been entrusted to the italian canadian team that is actually in charge of the France Canadian Lac StJean-Chicoutimi study. In particular, the Italian team is led by Prof. Giulio Antoniol and Prof. Michele Ceccarelli Università del Sannio-Benevento while the Canadian team is led by Dr. Ettore Merlo Ecole Polytechnique–University of Montreal. All the data obtained in the GENS Project will be stored in a denominized form in a dedicated data base and the access keys will entrusted to institutional or professional figures not related to the study and for that unable to have access to the data.
Once eligible families with relatives in both sites have been identified, all subjects will be invited to participate in a phenotyping session after an overnight fast. During this session participating subjects will sign the inform consent and will complete a form to collect information on familial and personal history of cardiovascular diseases. They will be submitted to a medical exam and phenotyping clinical protocol, which will include blood pressure measurements after rest in supine and standing positions, blood samples for general biochemistry, lipid profile and DNA extraction, anthropometric measurements to assess weight, BMI, waist circumference and skinfolds, as well as impedance measurements to estimate total body fat, lean mass and water percentages.
In the days following the phenotyping session subjects will be interviewed by phone in order to collect information on environmental exposures of interest, such as lifetime smoking and alcohol drinking, diet, physical exercise, occupational exposures, family history of cardiovascular disease, medications, as well as selected psychosocial and socio-economic factors associated with cardiovascular disease, using a set of questionnaires developed ad hoc for the GENS project
Prof. Donatella Tramontano
Dpt BPCM
00393404826207
We believe that this study represents a unique opportunity to capture gene-environment interaction, studying a population with the same genetic background but exposed to different lifetime environmental conditions.
The Institutions involved in Italy and in Canada
Università di Napoli “Federico II” Naples
Dpt BPCM “L. Califano”
Prof. Donatella Tramontano
Prof. Giovanni De Simone
Prof. Gabriele Riccardi
Dr. Annamaria Kisslinger
Dr. Giuseppina Sparaneo
CHUM-Université de Montréal Montreal
Prof. Pavel Hamet
Prof. Johanne Tremblay
Dr. Javier Pintos
Dr. Francis Gossard
Università del Sannio Benevento
Dpt ASES
Prof. Filippo Bencardino
Prof. Michele Ceccarelli
Prof. Francesco Mancini
Ecole Polytechnique Montreal
Prof. Ettore Merlo
Prof. Giulio Antoniol
Università del Molise Campobasso
Dpt SpS
Prof. Nicola Ferrara
Dr. Giovanni Villone
Fondazione S. Maugieri Telese Terme
Dr. Francesco Cacciatore
Santa Cabrini Hospital Montreal
San Raffaele Science Park Milano
University of Toronto Toronto
CIRB (Centro interuniversitario di ricerca bioetica) Naples
IREB Montreal
[1] Risk factor is defined as a diagnosis of hypertension and/or dyslipidemia, diabetes and obesity.
Cardiovascular outcome is defined in this proposal as a medical diagnosis of coronary heart disease (such as angina and myocardial infarction), cerebrovascular disease, and/or congestive heart failure (with or without hypertension).
Secondary outcomes
a-Prostate cancer. First, its familial aggregation is the highest among cancers, pointing to a substantial genetic component. Hypertension, hyperinsulinemia and other components of metabolic syndrome are among the risk factors for prostate cancer. Environmental risks of prostate cancer include nutrition and oxidative stress, confirmed by epidemiological studies as well as recent in vitro work using benign prostate hyperplasia and PC cell lines with “a potential for crosstalk” between cancer and hypertension we proposed in past. Furthermore, PSA is a recognised biological marker for prostate cancer detection, suitable for familial studies. Both past history and prevalence of prostate cancer will thus constitute a significant secondary outcome in the present study.
b-Erectile dysfunction
According to the NIH 30.000.000 men only in the US are affected by erectile dysfunction (ED) (NIH consensus conference). In recent years, several studies showed the association of ED with many CVD risk factors, including diabetes, hypertension, smoking and abnormal lipids. It has been suggested that ED may be a clinical marker of coronary, peripheral and cerebrovascular disease as well as hypertension and diabetes.
In our study we will investigate the correlation between CVD and ED risk factors and the influence of environmental factors in a family based study .
