PCR–RFLP

Fig. 1 Map of Guangxi Bama centenarian mitochondrial genome. The Guangxi Bama centenarian mitochondrial DNA (mtDNA) is a 16,569 bp circular molecule that codes for seven (ND1, 2, 3, 4L, 4, 5 and 6) of the 43 subunits of complex I, one (cytochrome b, cyt b) of the 11 subunits of complex III, three (COI, II, and III) of the 13 subunits of complex IV, and two (ATPase 6 and 8) of 16 subunits of complex V. It also codes for the small and large rRNAs, as well as 22 tRNAs with the adjacent letters indicating the cognate amino acids. PH, P and OH denote the heavy strand promoter, the light strand L promoter and the heavy strand replication, respectively

The mt5178A/C polymorphisms were analyzed by PCR- restriction fragment length polymorphism (PCR–RFLP). The primers used were designed according to Guangxi Bama mtDNA. The primer sequences were 50-CCCACA- TAGGATGAATAATAGCAG-30 and 50-TTACCACGC- TACTCCTACCTATCT-30, amplifying a 465 bp fragment. PCR reaction content contained about 1 lg of genomic DNA, 19 PCR buffer (10 mM Tris–HCl pH 8.3, 1.5 mM MgCl , 50 mM KCl), 0.2 mM dNTPs, 0.2 lM each pri- 2 mer, and 0.5U Taq DNA polymerase (Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). The PCR reaction conditions were the same as that for D-loop SNP described above. The PCR product was digested with the AluI restriction enzyme and then genotyped in 3.0 % agarose gel.

entire mitochondrial genome (Table 2). The sequences were assembled using the Phred-Phrap-Consed software.

Analyses of mitochondrial D-loop SNPs

Single nucleotide polymorphisms (SNP) of mitochondrial D-loop (mt-D-loop) were analyzed by direct sequencing of PCR products. Mt-D-loop regions are amplified using

Nucleotide position Alleles Locus Nucleotide change Amino acid MT-DLOOP A73G 73 A–G Noncoding MT-DLOOP A263G 263 A–G Noncoding MT-RNR2 A2076G 2076 A–G Noncoding MT-ATP6 A8860G 8,860 A–G Thr–Ala MT-ND4 G11719A 11,719 G–A Gly–Gly MT-CYB C14766T 14,766 C–T Thr–Ile MT-CYB A15326G 15,326 A–G Thr–Ala

mt146 100 a a 80 b 90-110 80-89 60 10-79 40 a 20 b b a a a 0 T C A )%( egatnecreP

described in the Materials and Methods. All ten Bama centenarians’ full mtDNAs were deposited in the GenBank database and their accession numbers were listed in Table 3. The full mtDNA of Bama centenarian was a double-stranded circular DNA molecule of 16,569 bp, encoding a total of 37genes and its structure map is shown in Fig. 1. Of the 37 genes, 13 were for polypeptides, 22 for transfer RNA and two for the small and large subunits of ribosomal RNA. The full mtDNA sequence revealed four haplogroups, i.e. M*, F1, D* and D4, in the ten Bama centenarians (Table 3). Compared with Cambridge Refer- ence Sequence, we found seven novel variations in the ten Bama centenarians’ mtDNA. Of these seven nucleotide variations, four do not change amino acids, including two in the D-loop region, one in the RNR2 gene and one in the ND4 gene, while the remaining three variations lead to a replacement of amino acids, including 8860 A/G (Thr/Ala) in the ATP6 gene, the 14766 C/T (Thr/Ile), and 15326 A/G (Thr/Ala) in the CYB gene (Table 4).

Fig. 2 Point mutation distribution at the mt146 in different age groups. Different letters on the bars indicate a significant difference between two groups (P\0.05) whereas the same letters indicate a lack of statistical significance (P[0.05). In the T genotype, n = 76, 58 or 99 for the 90–110, 80–89 or 10–79 years old groups, respectively. In the C genotype, n = 36, 15 or 22 for the 90–110, 80–89 or 10–79 years old groups, respectively. In the A genotype, n = 1, 2 or 4 for 90–110, 80–89 or 10–79 years old groups, respectively

MtDNA D-loop polymorphism analyses

direct sequencing of PCR products. Twenty-six variations were found in the 469 bp D-loop region of mtDNA from the 313 Bama individuals, of which only the variation at the mt146 was associated with longevity. The mt146T had a significantly lower frequency in the 90–110 years old group than that in the two younger (the 80–89 and 10–79 years old) groups (P \ 0.005) (Fig. 2). On the other hand, the mt146C occurred at a significantly higher frequency compared with the two younger groups (P \ 0.005) (Fig. 2). There were no significant differences for the mt146A genotype among three age-groups (P [ 0.005) (Fig. 2). The remaining 25 variations showed no significant difference among the three age-groups (P [ 0.05) (Table 5). The levels of systolic blood pressure as well as the total cholesterol and low density lipoprotein were significantly higher in those with the mt146C than in those with the mt146T genotype in the 90–110 years old group (P \ 0.05) (Fig. 3). Moreover, in the 90–110 years old cohort, the levels of triglyceride were much higher in those with the mt146C than in those with the

Statistical analyses

Alleles were evaluated for deviation from Hardy–Wein- berg equilibrium. Statistical analyses were performed using SAS statistical software version 8.02. The Pearson v2 test was used to analyze the genotype frequencies among the three age-groups. Serum parameters were analyzed using the least square means calculated from the general linear regression model (GLM) procedure.

Results

Full mitochondrial DNA sequence in Guangxi Bama Centenarians

The full mtDNA samples of 10 Guangxi Bama centenari- ans were sequenced using the PCR-based approach as

Table 5 Mitochondrial D-loop SNPs in Guangxi Bama populations

Locus SNPs 90–110 80–89 10–79 (n = 113) (n = 75) (n = 125) 282 W T 99.12 89.33 96.00 M1 A 0.88 8.00 3.20 M2 C 0.00 2.66 0.80 298 W C 97.35 94.67 92.80 M1 T 2.65 1.33 4.00 M2 A 0.00 4.00 3.20 310 0 28.32 42.67 34.40 INS C 71.68 57.33 65.60 311 0 85.84 90.67 80.80 INS C 14.16 9.33 19.20 318 0 0.88 9.33 1.60 INS C 99.12 90.67 98.40