the control group (P=0.31). The frequency of allele 9.3 was highest in both LLI and controls, consistent with previous reports. However, the association between TH01 and longevity observed in Italians was not replicated in German samples. The allele frequencies observed in Germans differed considerably from those observed in Italians, particularly with respect to allele 9.3. We observed a decreasing West-East and North-South cline across Europe for this allele, which had not been previously detected. This suggests that population-specific effects may explain the discrepant TH01-longevity association results obtained in Germans and Italians. Therefore, it is important to consider population genetic data when conducting association studies involving TH01.Either group (P40.05). When the data were analyzed, excluding smokers and individuals with high blood pressure, the results for the primary and secondary endpoints of the study were similar to those obtained considering all individuals. The results for the primary endpoint are shown in Table 3 and for detailed subgroup analyses in the Supplementary Material. For our primary endpoint, 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 centenarians). The result was again non-significant (relative LL frequency in The TH01 allele distribution differed considerably between Germans and Italians (Table 4), with the former exhibiting higher frequencies of alleles 9.3 and 10 while in the latter allele 6 is more common. However, our dataset is consistent with that obtained from a large representative sample from Germany, and the allele distribution described by Tan et al3 is in accordance with other data derived from Italy (Table 4). These findings suggest population-specific differences and ascertain the accuracy of the obtained data in both our and the Italian study.3 Table 1 shows genotype distribution of grouped TH01 STR alleles in German long-lived individuals and younger controls. 

The TH01 STR locus exhibits no significant influence on the ability of attaining exceptional old age in Germans. In a previous study, TH01 was reported to be associated with longevity in Italian men.1 A statistically significant decrease in the frequency of long alleles (Z9) was observed in centenarians compared with younger males from the northern (26 centenarians versus 49 controls) and southern (27 centenarians versus 113 controls) parts of the country. This finding would indicate a potential survival disadvantage for LL carriers. On the other hand, a later study on an extended and updated version of the original De Benedictis data described an increase in carriers of allele 9.3 in centenarians (n210) versus younger individuals (n755).3 This result suggests a beneficial effect of 9.3, a long allele, which seems to contradict the earlier finding.1 However, in our collections, there was no evidence for an association between long alleles of TH01, or specifically allele 9.3, and old age (Tables 1 and 2). The first association reported in Italians might reflect a potentially false-positive result caused by the very small sample sizes examined.1 The discrepant finding between Italians as reported by Tan et al3 and Germans (our study) could be due to a population-specific effect. Allele 9.3 is much less common in Italians (22%, Table 4) than in Germans (34%, Table 4). This remarkable frequency difference is consistent with other published data from the same populations (Table 4). The distribution of allele 9.3 differs remarkably among European populations, displaying a geographic gradient of decreasing frequency from West to East and North to South (Figure 2). If the TH01 STR is selectively neutral, the observed cline could be a consequence of past migrations or random genetic drift. But since the STR may regulate gene transcription in an allele-specific manner21 and may, therefore, be phenotypically relevant, the geographic allele distribution might very well reflect the adaptation to different environments and selective pressures. More detailed functional studies together with population genetic analyses are necessary to clarify this question. 

Taken together, our results do not provide evidence for the involvement of the TH01 locus in the modulation of human longevity in Germans. However, this finding does not rule out the intriguing possibility that such an association may exist in southern and/or eastern European populations. Furthermore, it highlights the need to take into consideration population genetic data when dealing with association studies. The lack of the TH01 longevity association in the investigated German sample means that at least based on our current knowledge, the forensic application of this robust and widely used marker is still legitimate.