aP < 0.05 for homozygotes more frequent in centenarians than in controls.

DISCUSSION

There is plentiful evidence for a direct role of HLA-DR alleles in differential susceptibility to a vast array of pathologies, in particular autoimmune diseases. The most classic examples include the association of DR4 and DR1 alleles with rheumatoid arthritis and of DR3 with systemic lupus erythematosus (22). The fact that these associations are found in a range of diverse ethnic groups, notwithstanding the various specific haplotypic combinations of polymorphic alleles extending over the whole region, strongly argues for a causal role of the implicated HLA-DR alleles. The penetrance of HLA-DR genotypes in disease phenotypes is commonly found to depend on sex (23). The influence of HLA-DR alleles on insulin-dependent diabetes mellitus (IDDM) is age- dependent (24). Furthermore, functional in vitro studies along with site-directed mutagenesis and sequencing have allowed in some instances the definition of the point mutations responsible for the different properties of certain alleles or subtypes (25,26). Among the numerous hypothetical mechanisms invoked to explain HLA class II disease associations, recent work has shown that signalling through HLA class II molecules mediates pathways leading to apoptosis (27).

This study presents the largest sample of longevous individuals ever genotyped for HLA-DR alleles, totalling 533 centenarians and 229 nonagenarians. Our selection criteria are original, in that they are designed to allow differentiation between cases of isolated longevity in one generation, represented by individual centenarians, and cases where there is evidence of familial aggregation in the same generation, represented by longevous siblings. The inclusion threshold of 95 years for women and 90 years for men in sibships was selected to balance the gender differential in longevity—there are roughly as many women over 95 as there are men over 90 in France. Within the longevous populations, four groups were further distinguished, according to sex and familial status, since (i) there were a priori grounds to expect sex-related influences; and (ii) centenarians and nonagenarian sibling-pairs had been recruited following distinct criteria. For the control group, we gathered genotypic data generated at seven different immunology centres corresponding to seven regions of France, totalling a population of 2950 adults. In spite of occasional regional fluctuations in certain allele frequencies, there was no significant heterogeneity within the control populations. In contrast, a significant heterogeneity among the HLA-DR allele distributions appeared among the five popula- tions including the controls and longevous groups. This provided a

In view of the implication of DR3 and DR4 alleles in autoimmune diseases, it is interesting that we did not find a significant effect of these alleles on longevity. DR3 has been proposed as a ‘male longevity factor’, especially within the A1B8DR3 haplotype (12,28)—otherwise associated with autoimmune disorders. A clue to this apparent paradox may come from our finding that DR3 is increased in longevous males from sibships (13.4%) whereas it is decreased in male centenarians (6.7%) relative to controls (11.0%). Very advanced age (>99) and status of familial aggregation regarding longevity had not been discriminated for in previous studies.

Therefore, in order to assess the contributions of individual alleles to longevity and their putative interaction with sex in this contribution, a log-linear model was implemented comparing either centenarians or nonagenarian siblings with adult controls for each allele. The allele-specific effects reached statistical significance for three alleles, namely DR7, DR11 and DR13, which made the largest contributions to the c 2 test for heterogeneity. The interaction with sex was significant for DR7 (elevated in longevous men, whether centenarians or siblings) and for DR11 (higher in women from sibships, unchanged in women centenarians). DR13 showed no interaction with sex, as its frequency was increased in centenarians of both genders.

We found a significant increase of DR7 in longevous men. DR7 appears to be a risk factor for CMV infection, either in renal transplant recipients (29) or in AIDS patients (30), and for chronic HBV infection (31). DR7 has been associated with the long-QT (LQT) syndrome, a polygenic electrocardiographic disorder char- acterized by syncope and fatal ventricular arrhythmias (32). While women are more affected than men by the LQT syndrome, the association was found only in men. Intriguingly, DR7 was positively and DR2 negatively associated with such disrelated diseases as LQT syndrome and HBV infection. HLA-DR7 has also been found as an additional risk for celiac disease in three independent populations from Spain, France and Ireland (33–35). Of note, though, celiac disease affects mainly women and, more importantly, the primary association comes from HLA-DQ rather than HLA-DR alleles (36). However, the fact that DR7 emerged in yet another population— from Germany—as the only class II allele significantly associated with Crohn’s disease (37), a pathology of the digestive tract like celiac disease, adds support to a direct involvement of this allele. Finally, DR7 has been associated with a chronic B cell lymphocyto- sis in French women (38). HLA-DR7 has proven ‘protective’ in two instances: against gold salt toxicity in rheumatoid arthritis treatment (39) and against disease progression in psoriatic arthritis (40).