Gene/longevity association studies were conducted at four autosomal loci (REN, THO, PARP, SOD2). The study included subjects older than 100 years and younger subjects matched for sex and geographic area (northern and southern Italy). The markers, located within their respective genes, were HUMREN4; HUMTHO1; HUMPARP; and SOD2(C/gt)845nt T. Multiallelic polymorphisms were recoded as diallelic according to allele size and frequency patterns. A significant loss of LL homozygous genotypes was found at the THO locus in male but not in female centenarians with respect to matched controls. However, no significant difference was found between case/control genotypic frequencies at REN, PARP, and SOD2 loci. The data is consistent with an association between the THO locus and longevity. Four autosomal genes were chosen as candidate loci: renin (REN), tyrosine hydroxylase (THO), poly(ADP-ribose) polymerase (PARP), and superexidodismutase 2 (SOD2). DNAs were extracted from blood buffy-coats following standard procedures. The genotypic pools of extremely elderly individuals and younger people from the same population were compared. The study revealed that the REN gene codes for renin, an aspartylprotease which catalyses the first step of the biosynthetic cascade leading to angiotensin II. An (acag)n repeat polymorphic region lies in intron 7 (HUMREN4 marker). The THO gene codes for tyrosine hydroxylase, the rate-limiting enzyme for synthesis of catecholamines. By alternative splicing of the THO transcript, four types of mRNA are produced. Specifically, in intron 1, an (aagt)n repeat polymorphism (HUMTHO1) is located. However, the data is consistent with an association between the THO locus and longevity. The PARP gene codes for a zinc finger nuclear enzyme which is strongly activated by DNA breaks and promotes DNA repair. PARP activity is positively correlated with species-specific life span in mammalian species. A (gt)n repeat polymorphism lies in exon 1. The SOD2 gene is a manganese-containing enzyme which is coded by a nuclear gene (6q25 chromosom) and is translationally imported into the matrix. Here, SOD2 rapidly converts superoxide radicals into H2O2 which are further detoxified by catalase and glutathione peroxidase. A structural t to c substitution in the mitochondrial targeting domain has recently been described which changes valine to alanine, thus modulating the efficiency of mitochondrial transport.allele sizes 10, 11, and 12 was statistically significant in southern Italy (P = 0.004 with α = 0.017) and had borderline significance in northern Italy (P = 0.027). No significant differences were found at the REN, PARP, and SOD2 loci. The genotypic analysis of individuals selected for longevity and younger people matched for sex and geographic area showed that the THO genotypic pools attained the same pattern in northern and southern Italian centenarians by means of hypothetical selection affecting survival. The aim of this study was to test whether the variability of four genes controlling fundamental metabolic pathways is associated with human longevity. The candidate loci tested in this study are not markers of longevity except for the THO locus.With respect to controls in both southern and northern Italy, differences in genotypic numbers and relative frequencies of the SOD2 (C/T) polymorphism are evident (Table 2), although statistical significance is borderline in northern Italian males when adjusting for multiple comparisons. In any case, we agree with Rothman28 that reducing the type 1 error for null associations increases the type II error for those associations that are not null. On the other hand, it is well known that the number of males who survive to very old age is much lower than that of females. If a certain genotype is unfavorable to longevity only (or primarily) in males, a gender gap in longevity should ensue. Our results suggest that the THO locus could be one of those which correlate with gender effect in longevity.

Table 4 shows genotypic numbers and relative frequencies of the SOD2 (C/T) polymorphism by sex and age group among controls and centenarians from North and South Italy. The finding that the HUMTHO1 marker is associated with long life expectancy and that such an effect is sex-related gives rise to two questions. Could a tandem-repeat polymorphic region affect life expectancy? Why does such an effect occur in males but not in females? As to the first question, HUMTHO1 lies in intron 1. It was proposed7 that secondary structures of the first intron and between first and second introns could affect THO alternative splicings. We might speculate that the variability of HUMTHO1 may play a role in the efficient production of spliced mRNAs. Indeed recent data has shown that the biological role of VNTR alleles of different size may be different.29,30 On the other hand, since HUMTHO1 and INS-VNTR markers are in linkage-disequilibrium and INS-VNTR allele frequencies show a sex-specific age-related trend,31 it may also be that sex-specific age-related variations of the genotypic pool embrace the entire DNA region comprising both THO and INS loci. In our samples at present, we are analyzing further markers occurring in the (11p15.5) DNA region to clarify this point. As to the second question, we must consider that the effect of a gene on a multifactorial trait depends on the physiological background in which the gene is expressed. Therefore, if the age-related physiological scenario changes in males and females differently, the effect of a certain gene on survival could vary between the sexes.

In conclusion, the data here reported shows that the genotypic pools at THO locus differ between male centenarians and male controls, with LL homozygous genotypes being very rare in centenarians. Furthermore, the specificity of the positive association found at the THO locus is validated by the negative findings at REN, PARP and SOD2 loci.