It should be noted that the GD method, as it has been applied in the present study, is potentially subject to two possible sources of bias. The first is that we recruited participants from a wide part of Italy (northern and central Italy), but we had to use demographic data referred to the whole Italian population, because a more restrictive and preferable set of data referred to northern-central Italy was not available in demographic databases. The second pos- sible source of bias is the contribution of migration flows which, when present, could violate the assumption of equal initial gene frequencies in different birth cohorts, required by the GD approach (28). However, in our study we included only northern and central Italian participants whose families were from the same geographic area (i.e., northern and central Italy) up to the third generation; consequently, in our opinion, the bias due to migration was likely minimal, even considering that in Italy the migration from south to north was quite rare before the middle of the last century. Furthermore, it should be noted that the effect of internal migration in Italy could hardly affect the application of the GD method to the TNF(A/G)-308 locus, given that allele frequencies at this locus do not vary among different areas of Italy. In fact, available data do not suggest a frequency variation of TNF(A/G)-308 alleles between northern and southern Italy, and a recently published article (39) reports exactly the same allele frequency distribution in 216 healthy controls from Northern Italy and 146 healthy controls from Southern Italy.

*Allelic combinations included in Caucasian conserved extended haplotypes in the MHC region (40).

the MHC region (40). TNF ¼ tumor necrosis factor.

TNF ¼ tumor necrosis factor.

ratio test to the data of Table 3 and found p ¼ .0027, thus confirming association between the TNF genomic region and age class. Due to the number of variables with respect to the sample size, it does not make sense to compare the frequency of each single haplotype among the age classes; however, it can be seen from Table 3 that the frequency of haplotypes including the A allele tends to decrease as the population ages and survival selection operates.

D ISCUSSION

The results of the present study indicate that the genotype at the TNF(A/G)-308 locus affects the probability of reaching extreme old age in a gender-specific manner. By using two different approaches both based on the addition of demographic to genetic data (Figure 1 and Table 2) we show that, in men, the presence of allele A in the genotype is unfavorable to attain longevity: The survival function is lower in allele A carriers than in noncarriers (Figure 1); the frequency of Aþ genotypes decreases with increasing age (Table 2). This observation is in agreement with the well- known association of the TNF(A/G)-308 polymorphism, and in particular of the A allele, with major age-related diseases, for example asthma, obesity, rheumatoid arthri- tis, systemic lupus erythematosus, and insulin resistance (14–20). From this perspective, the A allele is a frailty allele,

The haplotype data reported in Table 3 confirm that the entire TNF region is associated with the age of the sample group. Indeed, the whole distribution of haplotype frequen- cies differs among the three age classes (p ¼ .0027). This

Braun N, Michel U, Ernst BP, et al. Gene polymorphism at position -

of the tumor-necrosis-factor-alpha (TNF-alpha) in multiple sclerosis and its influence on the regulation of TNF-alpha production. Neurosci Lett.

;

:

–

Wilson AG, Symons JA, M

owell TL, M

evitt HO, Duff GW. Effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation. Proc Natl Acad Sci U S A.

;

:

–

Kroeger KM, Carville KS, Abraham LJ. The -

tumor necrosis factor-alpha promoter polymorphism effects transcription. Mol Immu- nol.

;

:

–

Danis VA, Millington M, Hyland V, Lawford R, Huang Q, Grennan D. Increased frequency of the uncommon allele of a tumour necrosis factor alpha gene polymorphism in rheumatoid arthritis and systemic lupus erythematosus. Dis Markers.

;

:

–

Chagani T, Pare PD, Zhu S, et al. Prevalence of tumor necrosis factor- alpha and angiotensin converting enzyme polymorphisms in mild/ moderate and fatal/near-fatal asthma. Am J Respir Crit Care Med.

;

:

–

Witte JS, Palmer LJ, O’Connor RD, Hopkins PJ, Hall JM. Relation between tumour necrosis factor polymorphism TNFalpha-

and risk of asthma. Eur J Hum Genet.

;

:

–

Herrmann SM, Ricard S, Nicaud V, et al. Polymorphisms of the tumour necrosis factor-alpha gene, coronary heart disease and obesity. Eur J Clin Invest.

;

:

–

Pihlajamaki J, Ylinen M, Karhapaa P, Vauhkonen I, Laakso M. The effect of the -

A allele of the TNF-alpha gene on insulin action is dependent on obesity. Obes Res.

;

:

–

Jaquet D, Tregouet DA, Godefroy T, et al. Combined effects of genetic and environmental factors on insulin resistance associated with reduced fetal growth. Diabetes.

;

:

–

Nicaud V, Raoux S, Poirier O, Cambien F, O’Reilly DS, Tiret L. The TNF alpha/G-

A polymorphism influences insulin sensitivity in offspring of patients with coronary heart disease: the European Atherosclerosis Research Study II. Atherosclerosis.

;

:

–

Antonicelli R, Olivieri F, Cavallone L, et al. Tumor necrosis factor- alpha gene -

G > A polymorphism is associated with ST-elevation myocardial infarction and with high plasma levels of biochemical ischemia markers. Coron Artery Dis.

;

:

–

Wang XY, Hurme M, Jylha M, Hervonen A. Lack of association between human longevity and polymorphisms of IL-

cluster, IL-

, IL-

and TNF-alpha genes in Finnish nonagenarians. Mech Ageing Dev.

;

:

–

Ross OA, Curran MD, Rea IM, et al. HLA haplotypes and TNF polymorphism do not associate with longevity in the Irish. Mech Ageing Dev.

;

:

–