precursor protein and is the only CNDP1 variant known to influence the enzyme activity in serum, with the exception of the rare null allele identified in the present study. The 5-leucine allele codes for a reduced activity of carnosinase and is thought to act as a recessive allele, with a strong functional impact only in the homozygous state. Previous studies by us (Janssen et al., 2005), independently confirmed by others (Barry Freedman, personal communication), showed that homozygosity for the 5- leucine allele protects against the development of nephropathy in diabetes mellitus. Considering the well-established cyto- protective effects of carnosine, we hypothesised that a similar beneficial effect would be likely with regard to longevity or the risk of developing cardiovascular disease. However, there was no significant difference between centenarians or CHD patients and their respective controls in terms of the frequency of neither the 5-leucine allele itself nor of homozygotes for it. Fisher’s exact test showed a difference in allele frequency spectrum between the longevity target and control groups which reached statistical significance (P = 0.021), owing to a higher frequency of the 7-leucine allele among centenarians. There is no conceivable biological explanation for a specific impact of this allele on the likelihood of reaching older age, and sequence analysis of the whole CNDP1 coding region did not reveal a genetic variant in linkage disequilibrium with the 7-leucine allele that could be assumed to influence carnosinase activity. The association may therefore be spurious.

Statistical analysis revealed no significant deviation from Hardy–Weinberg equilibrium in any of the four samples (all P > 0.1). A modest difference was observed between the allele frequency spectra of the longevity target and control group (Fisher’s exact P = 0.021), but not between the two KHK samples (Fisher’s exact P = 0.241; see Table 1). There was no significant difference between the target and control groups in the frequency of the 5-leucine allele or the proportion of individuals homozygous for the 5-leucine allele.

Since breakdown of carnosine in the bloodstream is mainly determined by serum-carnosinase whereas reduced activity of this enzyme, mediated by the shorter leucine repeat in the leader peptide, is not associated with longevity, a question mark must be put over the proposed longevity effect of carnosine as a nutrition supplement. The hypothesis that carnosine has this effect is mainly based on cell culture experiments and animal models. It should be noted that there is a big difference between carnosine metabolism in higher primates, who have serum carnosinase, and in other species which lack the complete

4. Discussion

We could not confirm an association of the functional leucine-repeat variant in the serum-carnosinase protein with either longevity or coronary heart disease. This variant is

supported by the EU-funded PREDICTIONS study on the identification of risk factors for the development of diabetic nephropathy (FP6-2004-LIFESCIHEALTH-5).

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During the course of our study, we identified three previously unobserved alleles in the CNDP1 gene. The rare 8-leucine allele is predicted to enhance protein secretion across the cell membrane and should therefore be associated with increased serum activity. The rare LAV duplication p.L13_V15dup, in contrast, may be functionally similar to the 5-leucine allele. Remarkably, we identified a novel frameshift mutation L17fsX20 that is predicted to completely abolish serum-carnosinase function. The mutation was found on 19 out of 2764 chromosomes investigated, representing a carrier frequency of approx. 1.4% in the German population. Complete absence of serum carnosine activity is thought to be the cause of carnosinaemia, a rare autosomal recessive trait with uncertain clinical relevance. No genetic analyses have yet been performed in affected individuals but it is possible that carnosinaemia is caused by homozygosity for L17fsX20 in some individuals, estimated to be found with a prevalence of approximately 1:20,000.

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