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 Genotype distribution of grouped TH01 STR alleles in German long-lived individuals and younger controls

Males Females Relative frequency Relative frequency Relative frequency Relative frequency controls (n¼275) LLI (n¼229) P-value controls (n¼187) LLI (n¼242) P-value SS 0.21 0.26 p ¼0.147 0.25 0.24 p ¼0.340 CCG CCG SL 0.55 0.46 0.47 0.53 LL 0.24 0.28 p ¼0.247 0.28 0.23 p ¼0.910 LL LL S/L 0.49/0.51 0.49/0.51 p ¼0.950 0.48/0.52 0.51/0.49 p ¼0.491 CCA CCA

Abbreviations: n, number of individuals; LLI, long-lived individuals; S, small allele (5, 6, 7, 8); L, long allele (9, 9.3, 10); p , P-value for overall genotype frequency comparison of controls and CCG LLI; p , P-value for frequency comparison of genotype LL against the other two genotypes SL and SS of controls and LLI; p , P-value for allele frequency comparison of controls and LLI. LL CCA

DISCUSSION

Table 2 Frequency distribution of carriers of the TH01 STR allele 9.3 in four age groups

controls suggest that 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 (n¼210) versus younger individuals (n¼755).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.

Carriers of allele 9.3 Age groups in years (n) Absolute frequency Relative frequency 19–59 (234) 126 0.54 60–75 (228) 141 0.62 96–99 (117) 70 0.60 Z100 (354) 189 0.53

Abbreviation: n, number of individuals.

Table 3 Results for comparison of grouped TH01 allele frequencies in subsamples of LLI and controls

p CCA p CCG p LL Analyzed subsamples Males Females Males Females Males Females All individuals 0.949 0.491 0.147 0.340 0.250 0.910 Non-smokers 0.831 0.432 0.224 0.553 0.237 0.876 Individuals free of 0.618 0.792 0.810 0.750 0.890 0.561 hypertension Non-smokers free of 1.000 0.913 1.000 0.916 1.000 1.000 hypertension

For abbreviations, see Table 1.

Table 4 Frequency distribution of TH01 alleles in Germans and Italians

Germans (n¼933), this Germans Italians Italians Allele study (n¼10285)22 (n¼965)a (n¼6045)22 4 0.00 0.00 0.00 0.0001 5 0.0021 0.036 0.00 0.0012 6 0.22 0.22 0.29 0.26 7 0.17 0.16 0.16 0.16 8 0.10 0.12 0.12 0.13 9 0.16 0.16 0.20 0.19 9.3 0.34 0.33b 0.22 0.26b 10 0.0086 0.017

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.

aData as listed in Table 1 of Tan et al.3 bOnly pooled data (allele 9.3 plus allele 10) are available.

0.28 0.33  ).3 0.36  0.33 0.27  37  0.31 0.3 (cid:129)3 0.3 (cid:129)1 0.32  0.35  0.22 0.32 ). 32 0.33   0.32 0.32   0.32   0.26 0.32 ).gé 0.26 0.23 ex  0.29 ).gs 0.18 .JE 0.29  0.21 

Figure 2 Map showing the frequency distribution of alleles 9.3 and 10 across Europe. For the comparison, allele 9.3 was considered together with the rare allele 10 as for many of the populations studied here no data are available for 9.3 separately. Data are from Huckenbeck et al.22

CONFLICT OF INTEREST