Molecular genetic studies on DNA polymorphism of the HLA class II 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 among both men and women are the highest in the world. In this study, we analyzed HLA class II alleles of Okinawan centenarians by the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method to clarify the presence of primary genetic factors in the major histocompatibility complex (MHC) region associated with human longevity. DRB1*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 explained by linkage disequilibrium with DRB1*1401 or vice versa. Furthermore, a tendency towards an increase was observed 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 longevity have become objects of medical and public interest (1, 2), few studies have been performed on the molecular basis of genetics to date, especially on human leukocyte antigen (HLA) in association with human longevity. In this report, we analyzed genetic polymorphism in HLA class II genes, focusing on DRB1, DQB1, and DQA1 alleles in Okinawan centenarians to clarify the possibility of the presence of primary genetic factors within the human MHC region associated with human longevity. Okinawa is the southernmost part of Japan and is well-known for one of the highest 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 data by the Ministry of Health and Welfare (6). Okinawa 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 consisted of 25 males and 95 females aged 100 to 110 years (mean standard deviation: 102.35 ± 1.9 years), and the controls consisted of 42 men and 87 women aged 20 to 75 years (63.551 ± 0.7 years). Physical check-ups and withdrawal of peripheral venous blood were carried out at their residence with informed consent. Consequently, only subjects who were in healthy conditions on clinical examinations were included. Both the centenarians and controls had been born and raised in Okinawa since their birth, and the controls were selected from the same vicinity of areas as the centenarians.

For HLA-DRB1, DQB1, and DQA1 genotyping, we used the PCR-RFLP method (8, 9) based on the digestion of PCR-amplified DNAs with allele-specific enzymes. 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 MgCl2, 10 mM Tris-HCl pH 8.4, 0.01% gelatin, 0.02% β-mercaptoethanol, 200 pM each of dATP, dCTP, dTTP, and dGTP, and 1 mM of each of the 5' and 3' primers in a total volume of 100 or 50 μl, was covered with 35 μl of mineral oil and subjected to 30 cycles of 1 min for denaturation, 1 min for annealing, and 2 min for extension by an automatic PCR thermal sequencer. After amplification, aliquots (5In this study, the allelic distributions of DRB1, DQB1, and DQA1 genes in centenarians and normal adults in Okinawa were compared. The results are summarized in Tables 1, 2, and 3. The frequencies of DRB1*10101 (x2=5.02, P=0.016, RR=4.300), DRB1*1201 (x2=3.56, P=0.032, RR=7.525), and DRB1*1401 (x2=12.93, P=0.0002, Pc=0.004, RR=4.146) were higher in centenarians than in normal adults. In contrast, the frequencies of DRB1*0403 (x2=4.69, P=0.027, RR=0.421) and DRB1*1302 (x2=6.18, P=0.007, RR=0.215) were lower in centenarians than in normal adults (Table 1). Only the positive association with DRB1*1401 remained significant after correction for multiple comparisons.

At the DQB1 locus, the frequency of DQB1*0503 was significantly increased in centenarians compared to normal adults (P=0.005, Pc=0.040, RR=2.508). The frequency of other HLA class I alleles in normal adults in Okinawa was similar to that of Japanese individuals reported previously. The deviations for DRB1*1401 and DQB1*0503 remained significant after correction for multiple comparisons.

Four alleles - DRB1*1401, DQB1*0503, DQA1*0101/0104, and DQA1*05 - were significantly associated with centenarians. The high frequency of DRB1*10101 in centenarians was consistent with previous findings. The low frequency of DQA1*0102 in centenarians may be explained by linkage disequilibria with DRB1*1302 and/or DQB1*0604. Similarly, the low frequency of DQB1*0302 in centenarians may be explained by the low frequency of DRB1*0403.

In conclusion, the study suggests that certain HLA class I alleles, such as DRB1*1401 and DQB1*0503, may be associated with longevity. However, further research is needed to determine the causal relationship between HLA class I genes and human longevity.I'm sorry, but there is no text enclosed in ``` ``` for me to proofread and improve. Could you please provide me with the text you want me to work on?