MATERIALS AND METHODS

University Hospital (Italy), as they were affected by AMI. The diagnosis of AMI was based on typical electrocardiographic changes and increased serum ac- tivities of relevant enzymes and confirmed by echocardiography and coronary angiography. The healthy, age-matched control group of 196 subjects was recruited amongst students or staff personnel who were checked and judged to be in good health based on their clinical history and on blood tests (complete blood cell count, erythrocyte sedimentation rate, glucose, urea nitrogen, cre- atinine, electrolytes, C-reactive protein, liver function tests, iron, proteins, cholesterol, triglycerides). The second control group consisted of 68 Sicilian centenarians, whose age ((cid:2)99 years) was verified by archival records at the City Hall and/or church registries. We paid particular attention to the concor- dance between reported age and personal chronologies (age of marriage and of military service for men; age of first and last pregnancy for women; age of children, among others). The characteristics of patient and control groups are reported in Table 1. The Sicilian ethnicity of the participants at the study was established by confirming that all four grandparents were born in Sicily; immigration and intermarriage have historically been rare. The University Hospital Ethics Committee approved the project, and an informed consent was obtained from each individual.

Concerning age-related controls, they were 117 women and 79 men; age range, 20–50; 39 were moderate smokers, and no other major cardiac risk factor was present. Concerning centenarians, they were 38 women and 30 men, (cid:2)99 years old, who did not have any cardiac risk factors or major age-related diseases (e.g., CHD, severe cognitive impairment, severe physical impairment, clinically evident cancer, or renal insufficiency), although some had decreased auditory and visual acuity. None of subjects referred any case of FMF in his family history.

10, corresponding to the carboxyl terminal of pyrin protein [9, 14]. Several studies about patients affected by FMF have identified the M694V mutation in a homozygous genotype as the most deleterious [14]. Although the molecular effects of these mutations are not yet known, they appear to be effective, even in the heterozygous state [15–18]. Hence, the presence of high carrier frequencies for these mutations in several geo- graphically related populations as well as above-normal levels of the acute-phase proteins C-reactive protein and serum amy- loid A in obligate carriers in FMF families [17, 18] suggests that people bearing pyrin mutations may have a selective advantage, possibly because of heightened resistance to patho- gen(s) [15, 16]. It has also been postulated that the low-grade inflammation in carriers, especially those carrying the M694V mutation [17], may well worsen clinical symptoms of immune- inflammatory diseases [17, 18]. Thus, we hypothesized that MEFV mutations may increase the risk of acute myocardial infarction (AMI) when interacting with ongoing atherosclerosis disease.

Blood specimens were collected in tripotassium EDTA sterile tubes, DNA- extracted, and processed for genotyping of three common FMF mutations: the M694V mutation, where G replaces A at position 2080 of the MEFV DNA (rs28940577); the M694I mutation, where A replaces G at position 2082 (rs28940578); and the V726A mutation, where C replaces T at position 2177 (rs28940479). The genotyping was performed by the amplification-refractory mutation system method according to published protocols [27], and random samples were genotyped again using restriction enzymes site analyses at the Heller Institute, Sheba Medical Center (Tel Hashomer, Israel), to confirm the results [20].

Allele and genotypic frequencies were evaluated by gene count and differ- ences in frequency among the groups. The (cid:3)2 test (2(cid:4)2 and 3(cid:4)2 tables, where appropriate) and ODD ratio (OR) with confidence interval (CI) were calculated. The data were tested for the goodness of fit between the observed and expected genotype frequencies according to the Hardy-Weinberg equilibrium (HWE) by (cid:3)2 test. In male patients and controls, we performed a logistic regression analysis to test the association of genotypes with AMI after adjustment for smoking habits, family history of CHD, and the presence of Type 2 diabetes, obesity, hypertension, hypercholesterolemia, and hypertriglyceridemia. The OR (with CI) was calculated as exponential of regression coefficient and its standard error.

three MEFV mutations commonly seen in the Mediterranean area, M694V, M694I and V726A [19, 20], in patients affected by AMI and age-matched controls from Sicily. First, to improve the power of our study, we selected genetically loaded cases  having early AMI onset ( 46 years) [21]. Second, we have studied a third group of subjects, consisting of a cohort of Sicilian centenarians, as in our previous studies, we have demonstrated that the achievement of longevity is facilitated by an anti-inflammatory status [22–25], and centenarians are a suitable control group for complex, age-related, inflammatory diseases, such as coronary heart disease (CHD) [1–4].

RESULTS

None of the Sicilian subjects carried the M694I or the V726A mutations. The proinflammatory M694V mutation, however,

TABLE 2. Frequency of Proinflammatory Alleles of Pyrin in AMI Patients, 196 Controls, and 68 Centenarians from Sicily

To date, the lifespan of MEFV mutation carriers or FMF patients has not been a matter of study. Langevitz et al. [26] have reported a similar incidence of CHD in patients with FMF treated with colchicine, their spouses, and the general popu- lation. It is well known, however, that before the introduction of colchicine, amyloidosis occurred in 60% of FMF patients over 40 years of age, being the main cause of death [9]. Moreover,