Molecular genetic studies on DNA U po1ymorpk;sm of the HLA class I1 associated with human longevity

'Research Center of Comprehensive Medicine, University of the Ryukyus, School of Medicine,

Department of Genetic Information. Division of Molecular Life Science, Tokai University School of Medicine

Okinawan Japanese are well known for their longevity; the population rate of centenarians in Okinawa is about 3.8 times higher than that of the whole Japan, where the average life expectancies both among men and among women are the highest in the world. In this study, we analyzed HLA class I1 alleles of Okinawan centenarians by the polymerase chain reaction-re- striction fragment-length polymorphism (PCR-RFLP) method for the pur- pose of clarifying the presence of primary genetic factors in the major histocompatibility complex (MHC) region associated with human lon- gevity. DRBI*1401, DQB1*0503, DQA1*0101=0104 and DQA1*05 were significantly increased in the centenarians. The significant increase of HLA- DQB1*0503 and/or DQA1*0101=0104 in the centenarians can be ex- plained by a linkage disequilibrium with DRBl*1401, or vice versa. Further, the tendency was observed toward increase with respect to DRB1*0101 and DRB1*1201. These data suggest that several alleles of the HLA-DRB1 and/or HLA-DQ genes are involved in human longevity.

Although biological contributions to human lon- gevity have become the object of medical and pub- lic interest (1, 2), few studies have been performed on the molecular basis of genetics to date, es- pecially on human leukocyte antigen (HLA) in as- sociation with human longevity.

In this report, we analyzed genetic polymorph- ism in HLA class I1 genes, focusing on DRB1, DQB1 and DQAl alleles in Okinawan centen- arians for the purpose of clarifying a possibility of the presence of primary genetic factor within the human MHC region associated with human lon- gevit y.

southernmost part of Japan and are well known for one of the longevity rates in the world (3). The average life expectancies of 76.7 years for males and 84.5 years for females in Okinawa prefecture were the highest for females and fifth highest for males in Japan (4), which has the highest average life expectancies in the world (5). Moreover, the population rate of centenarians was 22.14 per 100,000, which was about 3.8 times higher than that of Japan (5.87) in 1996, according to the data by Ministry of Health and Welfare (6).O kinawa was formed as an independent country between China and Japan until 1879, called the Ryukyu Kingdom, and was almost completely isolated

Material and methods

Subjects

One hundred and twenty centenarians and 129 normal adults in Okinawa prefecture were enrolled in this study. The subjects of centenarians consist of 25 males and 95 females, aged 100 to 110 years (meantstandard deviation: 102.35 1.9 years) and the controls of 42 men and 87 women, aged 20 to 75 years (63.551 0.7 years). Physical check-up and withdrawal of peripheral venous blood were car-

nature, 1 min for annealing, and 2 min for extension by automatic PCR thermal sequencer. After ampli- fication, aliquots (5 to 7 pl ) from the reaction mix- ture were digested with 2 units of restriction endo- nucleases for more than 3 hours at 37°C after ad- dition of appropriate incubation buffer.

ried out at their residence with informed consent. Consequently, only the subjects who were in healthy conditions on clinical examinations were included. Both the centenarians and the controls had been born and raised in Okinawa since their birth and the controls were selected in the same vicinity of areas as the centenarians.

Enzyme-treated reaction mixtures were investi- gated for cleavage and/or no cleavage patterns of amplified fragments by electrophoresis in 12% polyacrylamide gel. DNA fragments on the gels were detected by ethidium bromide staining. HLA class I1 alleles were defined on the basis of the gen- erated RFLP patterns (10).

Class II DNA typing

Genomic DNAs from centenarians and normal adults were purified using the proteinase K-phenol

For HLA-DRB 1, DQB 1 and DQAl genotyping, we used the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method (8, 9) based on the digestion of PCR- amplified DNAs with allele specific enzymes.

Statistics

The chi-square continuity table analysis with Yat- es' correction, the standard P-value (P)o f the Fisher's exact probability test and relative risk (RR), which were calculated from the cross prod- uct ratio of the entries in the 2x2 table, were used for assessment of the significance of results. Cor- rected P-values (P,)w ere calculated by multiplying P-values by the number of comparisons made for the DRB1, DQB1 or DQAl loci. A level of P,<0.05 was accepted as statistical significance.

Genomic DNA (200 to 300 ng) was amplified by the PCR procedure with 2 units of the Taq DNA polymerase. The reaction mixture, which contained genomic DNA, 50 mM KCl, 2.5 mM MgC12, 10 mM Tris-HC1 pH 8.4,0.01% gelatin, 0.02% "-40, 200 pM each of dATP, dCTP, dTTP, and dGTP, and 1 mM of each of the 5' and 3' primers in a total vol- ume of 100 or 50 p1, was covered with 35 pl of min- eral oil and subjected to 30 cycles of 1 min for de-

frequencies of HLA-OR81 alleles