On the basis of gene content, two groups of KIR haplotype have been defined as A and B. Common to both groups of haplotypes are the ‘‘framework genes’’: KIR3DL2, KIR3DL3, and KIR2DL4.14 Group A haplotypes contain not more than nine loci, which include KIR2DL1, 2DL3, 2DL4, 2DS4, 3DL1, 3DL2, 3DL3, 2DP1, and 3DP1. In contrast, the group B haplotypes are more diverse and have several genes coding activating receptors.

Several studies have associated KIR genes with disease susceptibility, immune responsiveness, and events following allogenic transplantation. Recent reports have implicated KIRs in affecting the outcome of hematopoietic stem cell transplan- tation.15 The genetic diversity of the KIR loci with respect to successful aging has been analyzed only in one study per- formed in the Irish population. Although, two KIR genes (2DS3, 2DL5) displayed an initial increased frequency in the aged group, the significance of this association was lost when repeated in a second cohort.16

To validate (in our homogeneous population) the gender- related associations between HLA-DRB1 genes and longevity observed in other Caucasoid populations, and to ascertain whether a particular repertoire of KIR receptors is associated with successful aging, we have evaluated HLA-DRB1 and KIR genotypes in centenarians and controls from Sicily.

Statistical analysis

Allele frequencies were evaluated by gene count, and 2(cid:1)2 tables were constructed to determine the statistical signifi- cance (chi-squared test with Yates correction) of differences in allele frequency for the HLA-DR polymorphisms and KIR genes between centenarians and controls. The p values ob- tained were multiplied for the number of alleles under study (Bonferroni correction), i.e., 14 for DR typing and 19 KIR typing. The data were tested for the goodness of fit between the observed and expected genotype values and their fit to Hardy–Weinberg equilibrium (HWE).

Materials and Methods

Subjects

We have genotyped 77 centenarians and 299 healthy con- trols for HLA-DRB1 and 44 centenarians and 57 healthy con- trols for KIR receptors=HLA ligands, all from Palermo and surrounding municipalities in western Sicily, Italy. The age of Sicilian centenarians was verified by archival records at the municipal offices and=or church registries. We paid particular attention to the concordance between reported age and per- sonal chronologies (age of marriage and of military service for men,age of firstand lastpregnancy for women,age of children, among others). This group did not have any cardiac risk factors or major age-related diseases (e.g., coronary heart disease, se- vere cognitive impairment, severe physical impairment, clini- cally evident cancer, or renal insufficiency), although some had decreased auditory and visual acuity, as would be expected.17 Most biochemical parameters, including cholesterol and tri- glycerides, were in the normal range. The healthy control group of 299 subjects was recruited amongst students or staff personnel who were checked and judged to be in good health on the basis of their clinical history and blood tests (complete blood cell count, erythrocyte sedimentation rate, glucose, urea nitrogen, creatinine, electrolytes, C-reactive protein, liver function tests, iron,proteins, cholesterol,triglycerides). Because immigration and intermarriage have historically been rare, the Sicilian ethnicity of all participants was established if all four grandparents were born in western Sicily. The study was ap- proved by the University Hospital Ethics Committee, and written informed consent was obtained from all participants.

Results

genes under investigation were consistent with those pre- dicted by the HWE. Table 1 shows the HLA-DRB1* allele frequencies in 299 controls and 77 centenarians. We found an increase of HLA DRB1*18 allele in male centenarians (p ¼0.0019), the increase remained significant after Bonfer- roni correction (p¼ 0.0266). Concerning the other alleles, no significant differences were observed between centenarians and controls (both in women and in men). A sample of subjects (38 healthy controls and 44 centenarians) was typed for HLA-B8, but there were no significant differences be- tween centenarians and controls concerning the HLA- B8,DR3 phenotype (data not shown).

Concerning KIR genes, Table 2 shows the KIR gene fre- quencies in 57 healthy controls and 44 centenarians. Only a significant decrease of the 2DP1 KIR gene in centenarians was observed (p ¼0.03), and it was no longer significant after Bonferroni correction. No significant difference was obtained stratifying the subjects by gender (data not shown). Table 3 shows HLA ligands frequencies observed in 57 healthy con- trols and 44 centenarians. No significant difference was ob- served in centenarians and controls for HLA ligands.

HLA and KIR typing

The salting-out method was used to extract the DNA, following the standard protocol.18 The DNA samples were

Table Allele Frequencies in Controls Women and Men 1. HLA-DRB1* 299 (171 128 ) and in Centenarians Women and Men from Sicily 77 (51 26 )