Note. Data are mean ± SD. *Therewere not statistically significant differ encesbetweenthegroupsonanyof thevariables.

Only the DNA of 10 centenarians was available to study re striction fragment length polymorphism (RFLP) TaqIpolymor phism in order to define the C4 and 21-hydroxylase (CYP 21; 21-0H) gene structure. The genomic DNA was digested with the restriction enzyme Taq I and hybridized with two 32P_Ia_ belledprobes,one beingspecific for the 5' end of C4A andC4B genes and the other for steroid21-0H gene. For a detailedde scription of RFLP TaqIpolymorphismpatternsseeref.22.

Table2.C4A,C4B,andBFAlleleFrequencies in Centenarians, ElderlySubjects,andYoung Controls*

Centenarians Elderly Young n=77 n= 89 n=235 Alleles n Frequency n Frequency n Frequency C4A A3 118 0.77 135 0.76 360 0.77 A2 13 0.08 14 0.08 38 0.08 A6 3 0.02 2 0.01 20 0.04 AQO 14 0.09 17 0.09 38 0.08 Others 6 0.04 10 0.06 14 0.03 C4B Bl 112 0.73 143 0.80 368 0.78 B2 11 0.07 17 0.10 46 0.10 BQO 26 0.17 16 0.09 49 0.11 Others 5 0.03 2 0.01 7 0.01 n=77 n = 81 n= 234 BF n Frequency n Frequency n Frequency S 113 0.74 113 0.70 340 0.73 F 34 0.22 45 0.28 105 0.22 S07 2 0.01 4 0.02 13 0.03 Fl 5 0.03 0 0 10 0.02

The complementactivity was testedvia the classicalpathway (CillO) using antibody-sensitized sheep red cells and veronal 2+, bufferedsaline(VBS) supplemented with Ca Mg2+, and 0.1% gelatinaccordingto Mayer (23) with slight modifications (24), and via the alternative pathway(AP50)employingunsensitized rabbit red cells and VBS containing 10 mM ethyleneglycolte tracetic acid (EGTA) and 7 mM Mg2+ according to published methods(25). C3 and C4levels were measuredimmunochemi callyby nephelometry.

Statistical analysis was performed using the chi-square test with theYate'scorrectionfor continuityand Bonferronicorrec tionfor the number of studiedalleles(26).Differencesbetween meanswere estimatedby unpaired,two-tailed,Student'st test.

REsULTS

Functional assaysperformed on serumsamplesof the subjects under study showedthat theyhad intactclassical and alternative complement hemolytic activities andplasmaconcentrations ofC3 andC4 withinthenormalrange(Table 1).Thefrequency distribu tion of C4A, C4B and BF allelewas similarin all three groups (Table 2).Thefrequencies ofC4Anullalleles weresimilar incen 2= tenarians versus healthy elderly persons (9.1% vs 9.5%; X = 2= 0.002;p .9)and versusyoungpeople(9.1% vs 8.1%;X 0.05, = p .8).An increase of C4BQO in the oldercohortwas observed: 2 centenarians versus old people (17% vs 9%, X = 3.9,p = .05), 2= centenarians versus young people (17% vs 11%; X 3.9;p .04), butthisdifference wasnotmaintained aftercorrection forthe number of alleles examined. The study at DNA level did not show, in centenarians, anyC4/21-0H genedeletionor otherchar acteristic gene patterns (data not shown). C3 allele frequencies were similarin the three groups (Table 3). The alleledistribution of C4, BF, and C3 alleles followed the Hardy-Weinberg equilib rium. Similarly statistical differences werenot seen whenthe age classes were comparedaccording to gender in centenarians and alsoinhealthyelderlyandyoungsubjects (datanot shown).

* Groups were compared with the chi- -square test. None of the comparisonswere statistically significant.

Table3.C3AllotypeFrequencyin Centenarians, ElderlySubjects, andYoungControls*

Centenarians Elderly Youngt = = n =75 n 29 n 213 Alleles n Frequency n Frequency n Frequency S 114 0.76 47 0.81 348 0.82 F 36 0.24 11 0.19 78 0.18 Genotype n % n % n % SS 43 57.3 18 62.1 144 67.6 FF 4 5.3 0 0.0 9 4.2 SF 28 37.4 11 37.9 60 28.2

*Groupswerecomparedwiththe chi-square test.None of the comparisons werestatistically significant. tThe rareallelesfoundinyoungbutnotin elderly and centenarians werenotconsideredfor statistical analysis (C3 rare allelesin = = 224youngsubjects, n 13;frequency .03%).

 

The widespread longevity in developed countries has ren dered it possible to study healthy centenarians (27,28); to our

ity genetic units for certain human diseases (35), the great major ity of individuals carrying such alleles are healthy and even in diseased cohorts, the linkage of this alleles with certain HLA class I and IT phenotypes renders it impossible to identify with certainty the genetic elements responsible for the disease. The partial defect of C4, a complement component of the classical pathway, which is potentially capable of damaging self tissues when locally activated by immune complexes, is known to be fully compatible with good health. Finally, the distribution of class ill alleles varies widely according to ethnic background, thence inferences on their relationship to pathology have to be considered within this framework Moreover, major histocompat ibility complex (MHC) allele and haplotype frequencies differ markedly even within different regions of the same country (36). Any demographic change in recent times like migration into or within a country or differential reproduction rates within ethnic subsets, rather than differences in fitness, could be responsible of observed different distributions. To partially overcome such con founding effects our studied cohorts were recruited with contri butions from Northern, Central, and Southern Italy, thence repre senting the Italian population as a whole.