Neuromed IRCCS, Pozzilli (IS) , Italy
Study design and endpoints
|“Moli-sani” (www.moli-sani.org) is a cohort study aiming at evaluating the risk factors (environmental, genetics, biomolecular) linked to chronic-degenerative disease with particular regard to cancer, cardiovascular disease and their intermediate phenotypes hypertension, diabetes, dyslipidemia, obesity and metabolic syndrome.
The Moli-sani study participants underwent anthropometric, biological and instrumental screening:
· Trained research personnel took BP and anthropometric measurements using methods standardized beforehand during preliminary training sessions. BP was measured by an automatic device (OMRON-HEM-705CP) 3 times on the not dominant arm and the last 2 values were taken as the BP. Measurements were made in a quiet room with comfortable temperature with the participants lying down for at least 5 min. Body weight and height were measured on a standard beam balance scale with an attached ruler with subjects wearing no shoes and only light indoor clothing. BMI was calculated as kg/m2. Waist circumferences were measured according to the NIH, Heart, Lung, and Blood guidelines.
· Subjects underwent electrocardiogram and spirometric test aimed to evaluate pulmonary diffusion capacity, gas diffusion and pulmonary volumes through plethysmography.
· Venous blood samples were obtained by clean venipuncture between 07:00 and 09:00 a.m. from participants who had fasted overnight and had refrained from smoking for at least 6 h. Biochemical analyses were performed in the centralized Moli-sani laboratory.
Glycaemia, cholesterol, HDL, LDL, C-reactive protein, D-Dimers and blood cell count were measured on fresh samples in the centralized Moli-sani clinical chemistry laboratories in the two recruiting centers. D-Dimer levels were measured in fresh citrate plasma, utilizing HemosIL, an automated latex immunoassay on IL coagulation System ACL9000. Quality control was maintained using internal laboratory standard in-house plasma pool. Inter and intra-day CV was 5.4% and 7.6%, respectively.
High sensitivity CRP was measured in fresh serum sample, using quantex CRP ultra sensitive immuno turbidimetric assay on ILab350.
Serum lipids and blood glucose were assayed by enzymatic reaction methods using an automatic analyzer (ILab 350). LDL-cholesterol was calculated according to Friedewald.
Blood cell counts including platelets, white and red blood cells, mean corpuscular volume (MCV) and haemoglobin (Hb) were performed by a Coulter LH Hematology analyzer (Beckman-Coulter).
Moreover, in the central laboratory of the European project BiomarCaRE the following biomarkers were measured on frozen samples: Apoliprotein A, Apoliprotein B, C-peptide, Creatinine, C-reactive protein, Cystatin C, Glucose, HDL, Insulin, LDL, Lipoprotein(a), N-terminal-pro B-type natriuretic peptide, Testosteron, Triglycerides, hs Troponin I, Vitamin D.
· Citrated plasma, EDTA plasma, serum, cellular pellet and urinary spots are stored in liquid nitrogen in a specific Biobank. DNA is going to be extracted and stored at -80°C
Each subject was asked to answer to three different types of questionnaires: anamnestic, food frequency and quality of life (FS36) questionnaires and underwent psychometric tests to evaluate any depressive status, stress response and attitude to suicide:
· The first questionnaire, beyond personal data, collected information on social status and childhood, physical activity, smoking habits, hormonal status in women, body weight history, inflammatory disease in the past year, antibiotics and influenza vaccine, hypertension, diabetes and dyslipidemia and relative dietary and pharmacological treatment, ischemic cardiovascular disease and relative treatment (type of medication, dosage and compliance) and possible hospitalization, pulmonary diseases (relative treatment and possible hospitalization), depressive status or anxiety with relative previous and current treatment, haematological disease, kidney disease, gastrointestinal disease, endocrinological disease, Alzheimer disease, tumours (malignant and benign), surgical intervention and radiological tests in the past five years (with indication of the Centres where they were performed).
Finally, the questionnaire presented a detailed pharmacological analysis registering each chronic or acute medication in the past 15 days, with indication of commercial name, type of packaging, dosage, frequency, compliance and clinical indication (people are asked to bring with them the GP prescription or the drug packaging of the medication they use to take).
· Dietary assessment. The European Prospective Investigation into Cancer and Nutrition FFQ was used to determine daily nutritional intakes consumed in the past year. This questionnaire was designed and validated in a multicenter study performed in 10 European countries, with the aim to evaluate the relation between diet and cancer. The questionnaire, computerized with tailor-made software, allowed researchers to interview participants in a fully interactive way, including illustrations of sample dishes of definite sizes or by reference to standard portion sizes. All the answers provided by the participants were registered in a database in real time. The NAF software (Nutritional Analysis of Food Frequency Questionnaires, National Cancer Institute, Milan, Italy) (17) was used to transform information about food composition into daily intake of food items (g/d), energy (kcal or kJ/d), and macro- and micronutrients (g or mg/d). Nutrient data for specific foods were obtained from the food composition database for epidemiological studies in Italy.
On the basis of the recorded data, each subject was provided with his own cardiovascular risk according to the cuore.exe algorithm elaborated within the Cuore Project by ISS.
A follow-up based on linkage with hospital discharge records, clinical records and mortality regional registry was performed at December 2011 (FUP1) with a median of 4.3 years.
· Validated end-points followed up: Deaths (with specific causes of death), fatal or Non-fatal AMI, cardiac revascularization, fatal and non-fatal stroke events, heart failure, atrial fibrillation, diabetes, cancer, neurodegenerative disease (ongoing).
· A second follow-up (at December 2019) is ongoing.
· At June 2015 re-examination at the cohort has stated.
Scientific advice / protocol assistance / communication with regulatory / competent authorities / ethics committees
The Moli-sani study was approved by the ethics committee of the Catholic University of Rome and is conducted under the supervision of both the Bioethics Institute of the Catholic University of Romethe and the Istituto Superiore di Sanità, Rome. All participants signed an informed consent before taking part in the study. This study was conducted in accordance with the Declaration of Helsinki.
The study has recruited, between March 2005 and April 2010, people aged ³ 35 living in the Molise region from city hall registries by a multistage sampling. First, townships were sampled in major areas by cluster sampling; then, within each township, participants aged ≥35 years were selected by simple random sampling. In particular, once sampled the index person, all the nuclear family members aged over 34 years were invited to participate: starting from the nuclear family structure, it is possible to build-up large family pedigree.
Exclusion criteria were pregnancy at the time of recruitment, disturbances in understanding or willingness, current poly-traumas or coma, or refusal to sign the informed consent.
From 2005, 24,325 participants were enrolled, with a participation rate of 70%.
- Women: N 12,623 (51.9%);
- Age 35-64yrs: N 18. 494 (76%); 65-74yrs: 4,087 (16.8); ≥75yrs: 1,744 (7.2%)
- Total Mortality: N 575 (2.4%)
- CVD events:1,367 (5.6%)
- Diabetes II Type: 369 (1.5%)
- Heart failure: 463 (2.1)
- Atrial fibrillation: 395 (1.8)
- Cancer (validation on going): 765 (3.1)
FUP2 (on going):
- Total Mortality: 1,208 (5.0%)
- Heart Failure: 862 (3.5)
- Atrial fibrillation: 526 (2.2)
Neurodegenrative diseases: 350 (1.4%)
For database management and statistical analysis, SAS system, version 9.4 (SAS Institute Inc., Cary, NC) is used
|Nº of subjects||24,325|
|Sex (%), women||12,623 (51.2%)|
|Age (SD), years||55.8 (12.0)|
|Sampling frame||Family-based random sample|
|Starting point to recruit sample||City registry (30 towns of Molise Region)|
|Follow-up||FUP1: 2005-2011 ended; FUP2: until 2019 ongoing|
|Samples||Citrated plasma, EDTA plasma, serum, spot urine samples (3hr morning urines), buffy coats, DNA (extraction on going)|