0.35 0.32 0.3 LLI controls 0.24 0.20 0.2 0.160.18 0.17 0.16 0.10 0.10 0.1 0.0010.003 0.011 0.0 5 6 7 8 9 9.3 10 ycneuqerf

TH01 typing

Five nanograms of genomic DNA from each individual, extracted from whole- blood samples, was subjected to a duplex PCR amplifying a TH01-specific STR fragment and the gender-determining amelogenin locus. Primers and PCR conditions were used as described previously.18 Amplification products were separated and detected on an ABIPrism3130 Genetic Analyzer with ROX500 standard fragments (all Applied Biosystems, Weiterstadt, Germany) in compar- ison with a self-made allelic ladder. Electrophoresis results were analyzed using the GeneMapper ID Software v3.2 with the respective self-designed bin set. Allele peaks were scored when the relative fluorescence units were between 50 and 3000.

Statistical analysis

The primary aim of the study was to try to validate the result of De Benedictis et al.1 Secondary end points of the study were other potential differences in the TH01 allele distribution between cases and controls, with a special focus on allele 9.3. All P values were two-sided and were considered significant if smaller than 0.05. For the primary end point, only one test was performed. Owing to the exploratory nature of the study with respect to the secondary end points, no adjustment for multiple testing was conducted.

Figure 1 Relative frequencies of ungrouped alleles of the TH01 STR in German LLI and younger controls.

Alleles were grouped according to the previous study of De Benedictis et al:1 small (S) – 5, 6, 7, 8; large (L) – 9, 9.3, 10. The alleles ‘S’ and ‘L’ are designated in the following as grouped alleles. The allele ‘10–1’ of De Benedictis et al,1 also called ‘10*’ by Tan et al,3 is referred to as ‘9.3’ in the forensic literature19 and in this study. Allele and genotype distributions between cases and controls were compared by applying Fisher’s exact test. Independence of the occurrence of allele 9.3 and other alleles in the same genotype was tested with a w2 goodness-of-fit test. This coincided with a test for Hardy–Weinberg equili- brium. Logistic regression was performed with a carrier status of allele 9.3 as outcome and age as continuous influence variable. The distribution of 9.3 carriers was also studied in four different age groups. Sample size and power calculations were done with BIAS for Windows 8.03. For all other statistical calculations the statistics program R was used (version 2.5.120). For tests for Hardy–Weinberg equilibrium the package ‘genetics’ was applied: HWE.exact for two alleles and HWE.chisq for more than two alleles (R package version 1.3.0, 2007).

LLI: 0.30, in controls: 0.21, P-value for comparison: 0.067). The frequency of the LL genotype was higher in German LLI than in controls, in contrast to the result in Italians.1

In our sample, the distribution of the ungrouped allele and genotype frequencies in cases and younger controls was very similar (Figure 1). An overall comparison of allele or genotype frequencies showed no significant difference between the two groups (alleles: P¼0.48, genotypes: P¼0.71).

Allele 9.3 was the most common one in Germans (Figure 1). Tan et al3 reported an increase in carriers of allele 9.3 in Italian centenar- ians versus younger controls. When 9.3 was analyzed separately against all other alleles in the German sample, no significant deviation between cases and controls was found (P¼0.24). In addition, there was no significant result of the increased presence of 9.3 with any of the other alleles in a heterozygote genotype (P40.05 for all other alleles). Furthermore, logistic regression analysis, in which the 9.3 carrier status was considered as outcome and age as continuous influence variable, showed no significant result (P¼0.73). Also, the frequency distribution of 9.3 carriers in the four examined age groups was very similar and appeared to fluctuate randomly (P¼0.16; Table 2). The frequency of carriers in the centenarian group was lower than in the younger age groups, in contrast to previous findings.3

Smoking and high blood pressure both affect mortality and ageing and may be associated with variation in the TH01 gene.7,12,13 To investigate the robustness of our results and to prevent confounding, we repeated the analysis considering only non-smokers (n¼470) and individuals free of hypertension (n¼550).

RESULTS

In the German samples, seven alleles were observed at the TH01 locus in both LLI and controls (Figure 1). No significant deviation from Hardy–Weinberg equilibrium was found in either group (P40.05).

When the data were analyzed excluding smokers and individuals with high blood pressure, the results for the primary and secondary end points of the study were similar to those obtained considering all individuals. The results for the primary end point are shown in Table 3 and for detailed subgroup analyses in the Supplementary Material.

For our primary end point, we applied the same study design as described in De Benedictis et al1 and grouped the alleles as small (S) or large (L). In De Benedictis et al1 it was shown that the LL genotype was significantly less frequent in male centenarians when compared with the other two genotypes, SL and SS. For a significance level of 0.05, our samples had a power of over 99 or 89%, respectively, to replicate the previous results observed for South Italian males and females.1 Despite sufficient power, the association between TH01 and longevity was not replicated in the studied German samples (P values for comparison of LL versus SL/SS: 0.25 for males, 0.91 for females; Table 1). Furthermore, we repeated our analysis for the subgroup of centenarian male cases (the German female cases were all centenar- ians). The result was again non-significant (relative LL frequency in